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1 MICROBIAL INACTIVATION AND ALLERGEN MITIGATION OF FOOD MATRIX BY PULSED ULTRAVIOLET LIGHT By AKSHAY KUMAR ANUGU A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE RE QUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Akshay Kumar Anugu
3 To t he tax p ayers of India and the United States of America
4 ACKNOWLEDGMENTS I would like to express sincere thanks and appreciation t o my advisory committee: Dr s Wade Yang ( major advisor ) Keith Schneider, Maurice Marshall and Bruce Welt for their invaluable support, suggestions and guidance pertaining to my research and matriculation in the Ph.D. program of the Department of Food Scie nce and Human Nutrition at the University of Florida. Second, I would like to acknowledge my family and friends who have always been there to uplift and offer me words of encouragement Moreover, I would also like to express my deepest gratitude to my lab mates Cheryl Rock, Senem Guner, Sandra Shriver, Xi ngyu Zhao, Bhaskar Janve, Syed Abbas (Ali), Ying Guo (Kelsey) Yiq iao Li (Chelsey) Jyotsna Nooji and Samet Ozturk for their continuous encouragement, moral support and advice. Last, I would also like to acknowledge Dr. Susan Percival, Mrs. Bridget Stokes Mr. Rob Pelick, Dr. Wlodzimierz Borejsza Wysocki and Dr Mi l e na Ramirez Rodrigues for their guidance and kindness pertaining to the use of their lab facilities and equipment. Also, I would like to thank the office staff of the Food Science and Human Nutrition Department: Ms. Marianne Mangone, Mrs. Rhonda Herring Ms. Shelia Parker Ms. Julie Barber, Ms. Carmen Graham Ms. Mary Ann Spitzer and Ms. Janna Underhill for taking care of me during the course of my academic matriculation
5 TABLE OF CONTENTS p age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREVIATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Justification of Study ................................ ................................ ............................... 16 Objectives ................................ ................................ ................................ ............... 17 2 LITERATURE REVIEW ................................ ................................ .......................... 19 Nonthermal Food Processing Technology ................................ .............................. 19 Pulsed UV Light ................................ ................................ ................................ ...... 19 Variables ................................ ................................ ................................ .......... 21 Sample Thickness ................................ ................................ ............................ 21 Treatment Time ................................ ................................ ................................ 23 Distance from Light Source ................................ ................................ .............. 23 Factors Affecting PUV Efficacy ................................ ................................ ............... 24 Pulsed UV Light Applicat ions ................................ ................................ .................. 28 Inactivation of Microorganisms ................................ ................................ ......... 28 Photochemical effect ................................ ................................ .................. 29 P hotothermal effect ................................ ................................ .................... 30 Photophysical effect ................................ ................................ ................... 32 Photodynamic effect ................................ ................................ .................. 32 Photoreactivation ................................ ................................ ....................... 34 Effect of PUV on Food Quality ................................ ................................ ................ 35 3 EFFICACY OF PULSED ULTRAVIOLET LIGHT ON ANTIGENICITY OF ISOLATED MILK PR OTEINS ................................ ................................ ................. 38 Background ................................ ................................ ................................ ............. 38 Materials and Methods ................................ ................................ ............................ 4 0 Equipment ................................ ................................ ................................ ........ 40 Experimental Design ................................ ................................ ........................ 40 Materials ................................ ................................ ................................ ........... 41 Allergen analysis ................................ ................................ .............................. 41
6 Gel electrophoresis and western blot ................................ ......................... 41 Indirect ELISA ................................ ................................ ............................ 42 Data Analysis ................................ ................................ ............................. 43 Results and Discussion ................................ ................................ ........................... 43 SDS PA casein ................................ ....................... 43 SDS PAGE and Western Blot for Whey Protein ................................ ............... 45 Indirect ELISA ................................ ................................ ................................ .. 48 Other Benefits ................................ ................................ ................................ .. 50 4 EFFECTS OF PULSED ULTRAVIOLET LIGHT AND HIGH HYDROSTATIC PRESSURE ON THE ANTIGENICITY OF ISOLATED EGG PROTEINS ............... 60 Background ................................ ................................ ................................ ............. 60 Materials and Methods ................................ ................................ ............................ 63 SDS PAGE and Western Blot ................................ ................................ .......... 64 Indirect ELISA ................................ ................................ ................................ .. 65 Statistical Analysis ................................ ................................ ................................ .. 66 Results and Discussion ................................ ................................ ........................... 66 SDS PAGE and Western Blot for PUV Treated Samples ................................ 66 Indirect ELISA for PUV Treated Samples ................................ ......................... 68 Cost Comparisons of PUV Treatment ................................ .............................. 69 Effect of PUV on Sample Temperature and Moisture ................................ ....... 70 Effect of High Hydrostatic Pressure ................................ ................................ .. 71 5 ANTIOXIDANT CAPACITIES AND HEALTH ENHANCING PHYTONUTRIENT CONTENTS OF SOUTHERN HIGH BUSH BLUEBERRY WINE COMPARED TO GRAPE WINES AND FRUIT LIQUORS ................................ ........................... 83 Background ................................ ................................ ................................ ............. 83 Materials and Methods ................................ ................................ ............................ 86 Southern Highbush Blueberry Wine Samples ................................ .................. 86 Determination of Antioxidant Capacity ................................ ............................. 86 Determination of Total Phenols ................................ ................................ ........ 87 Determination of Total Flavonoids ................................ ................................ .... 87 Determination of Total Anthocyanins ................................ ................................ 88 Statistical Analysis and Comparison ................................ ................................ ....... 89 Results and Discussion ................................ ................................ ........................... 89 Antioxidant Capacities, Total Phenols, Anthocyanins, Flavonoids of Southern Highbush Blueberry Wine ................................ .............................. 89 ORAC Comparison between Blueberry Wines and Grape Wines .................... 91 Comparison of Total Phenols and Anthocyanins among Blueberry Wines, Fruit Liquors and Grape Wines ................................ ................................ ..... 92 6 EFFECT OF PULSED UV LIGHT ON SACCHAROMYCES CEREVISIAE IN SOUTHERN HIGH BUSH BLUEBERRY WINE AND WHITE GRAPE WINE ....... 100 Background ................................ ................................ ................................ ........... 100
7 Materials and Methods ................................ ................................ .......................... 101 Materials ................................ ................................ ................................ ......... 101 Experimental Procedure ................................ ................................ ................. 102 Results and Discussion ................................ ................................ ......................... 103 Temperature ................................ ................................ ................................ ... 103 Inactivation of Saccharomyces cerevisiae in Blueberry Wine ......................... 103 Inactivation of Saccharomyces cerevisiae in White Grape Wine .................... 105 7 EFFECT OF PULSED UV LIGHT ON PHENOLIC, ANTIO XIDANT ACTIVITY, COLOR AND AROMA OF SOUTHERN HIGH BUSH BLUEBERRY WINE .......... 112 Background ................................ ................................ ................................ ........... 112 Materials and Methods ................................ ................................ .......................... 114 Southern Highbush Blueberry Wine Samples ................................ ................ 114 Determination of Antioxidant Capacity ................................ ........................... 114 Determination of Total Phenols ................................ ................................ ...... 115 Determination of Total Flavonoids ................................ ................................ .. 115 Determination of Total Anthocyanins ................................ .............................. 116 Gas Chromatography Mass Spectrometry Volatile Aroma Analysis ............ 117 Results and Discussion ................................ ................................ ......................... 117 Total Phenolics, Anthocyanins, Flavonoids and Antioxidant Capacity ............ 117 Color ................................ ................................ ................................ ............... 119 Volatile Aroma ................................ ................................ ................................ 119 8 CONCLUSIONS AND RECOMMENDATIONS ................................ ..................... 126 NOMENCLATURE ................................ ................................ ................................ ...... 128 LIST OF REFERENCES ................................ ................................ ............................. 130 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 143
8 LIST OF TABLES Table page 5 1 The ORAC values, total phenols (TP) anthocyanins and flavonoids of Southern highbush blueberry wine with and without sulfite addition or filtration. ................................ ................................ ................................ .............. 94 5 2 Comparison of the ORAC values among the red, Rose, white and blueberry wines ................................ ................................ ................................ .................. 95 5 3 Total phenol and total anthocyanin comparison among the red, Rose, white and bl ueberry wines and fruit liquors ................................ ................................ .. 97 6 1 Reduction of Saccharomyces cerevisiae in blueberry wine when treated with PUV at a dist ance of 6 cm from quartz window ................................ ................ 107 6 2 Reduction of Saccharomyces cerevisiae in white grape wine when tr eated with PUV at a dist ance of 6 cm from quartz window ................................ ......... 108 7 1 Effect of PUV treatment on lightness and hue of blueberry wine when treated at a distance of 6 cm from quart z window ................................ ........................ 124 7 2 Identified major volatile aroma compounds of blueberry wine when treated at a dist ance of 6 cm from quartz window ................................ ............................ 125
9 LIST OF FIGURES Figure page 3 1 SDS PAGE for whey proteins showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3 ) PUV treatment for 180 s ......... 51 3 2 SDS PAGE for casein showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3) PUV treatment for 180 s ......... 52 3 3 Western blot for whey protein showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3) PUV treatment for 180 s ......... 53 3 4 Western blot for casein showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3) PUV treatment for 180 s ......... 54 3 5 Indirect ELISA results for PUV treated isolated milk proteins indicating reduction in IgE binding after PUV treatment ................................ ..................... 55 3 6 Amin lactoglobulin ................................ ............................ 56 3 7 lactalbumin ................................ ............................. 57 3 8 s2 casein ................................ ................................ 58 3 9 s1 casein ................................ ................................ 59 4 1 SDS PAGE for egg proteins showing decrease in band intensities of major allergens afte r PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3) PUV treatment for 180 s ......... 73 4 2 Western blot for egg proteins showing decrease in Ig E binding of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, a nd (T3) PUV treatment for 180 s ......... 74 4 3 Indirect ELISA resu lts for PUV treated isolated egg proteins indicating reduction in IgE binding after PUV treatment ................................ ..................... 75 4 4 SDS PAGE for egg proteins showing band intensities of major allergens after HHP treatmen t. (M) Marker, (C) Control, (H1) at 4 o C for 5 min, (H2) at 4 o C for 15 min, (H3) at 4 o C for 30 min, (H4) at 21 o C for 5 min, (H5) at 21 o C for 15 min, (H6) at 21 o C for 30 min, (H7) at 70 o C for 5 min and (H8) at 70 o C for 15 min. ................................ ................................ ................................ ................ 76
10 4 5 Western blot for egg proteins showing decrease in IgE binding of major allergens after HHP treatment. (M) Marker, (C) Control, (H1) at 4 o C for 5 min, (H2) at 4 o C for 15 min, (H3) at 4 o C for 30 min, (H4) at 21 o C for 5 min, (H5) at 21 o C for 15 min, (H6) at 21 o C for 30 min, (H7) at 70 o C for 5 min and (H8) at 70 o C for 15 min ................................ ................................ ...................... 77 4 6 Indirect ELISA results for HHP treated isolated egg proteins in dicating increase in IgE binding after HHP treatment ................................ ...................... 78 4 7 Amino Acid sequence for ovalbumin ................................ ................................ .. 79 4 8 Amino Acid seq uence for chick en serum albumin ................................ .............. 80 4 9 Amino A cid sequence for ovotransferrin ................................ ............................. 81 6 1 Change in surface temperature of 5 ml blueberry wine when tr eated with PUV for 60 s at a dist ance of 6 cm from quartz window ................................ ... 109 6 2 Change in temperature of 10 ml blueberry wine at 2.5 mm distance from surface when treated with PUV for 60 s at a dist an ce of 6 cm from quartz window ................................ ................................ ................................ ............. 110 6 3 Change in temperature of 15 ml blueberry wine at 5 mm distance when treated with PUV for 60 s at a distance of 6 cm from quartz window ................ 111 7 1 GC MS chromatogram for volatile aroma of 5 s PUV treated blueberry wine. .. 121 7 2 GC MS chromatogram for volatile aroma of 25 s PUV treated blu eberry wine 122
11 LIST OF ABBREVIATION S ANOVA Analysis of variance CFR Code of federal regulations CMVS Color machine vision system D PPH 1, 1 diphenyl 2 picrylhydrazyl DNA Deoxyribonucleic acid EDTA Ethylenediaminetetraacetic acid ELISA Enzyme linked immune sorbent assay EPA Environmental protec tion agency FAO Food and agricultural organization FDA United States Food and Drug Administration FRAP Ferric reducing antioxidant potential GMP Good Manufacturin g P ractice HHP High hydrostatic pressure processing I R Infrared ORAC Oxygen radical scavenging activity PU V P ulsed ultra violet light SAS Statistical analysis system SD Standard deviation SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis USDA United S tates D epartment of A griculture UV/VIS Ultraviolet/visible
12 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy MICROBIAL INACTIVATION AND ALLERGEN MITIGATION OF FOOD MATRIX BY PULSED ULTRAVIOLET LIGHT By Akshay Kumar Anugu August 201 3 Chair: Wade Yang Major: Food Science and Human Nutrition Nonthermal processes have gained importance in recent years as a potential techno log y to replace or complement the tr aditional thermal processing of foods. Puls ed UV light (PUV) was shown to be effective in inactivating several microorganisms and mitigating antigens to ensure food product safety. Promising results about minimal damage to the food product quality can ensu re the application of PUV as a commercial viable nonthermal process. The major objective of this study was to evaluate the potential applications of PUV for different food matrices in inactivating microorganisms and antigens, while preserving the qual ity o f food such as micro nutrients, color and aroma. Milk is among eight major foods that are responsible for more than 90% of food allergies. Milk allergy is a major cause of transient food hypersensitivity in children. Egg allergy is the second most common f ood allergy, affecting 1.6% to 3.2% of young children. Fiv e ml lactoglobulin, lactalbumin and casein) and egg proteins were treated at a distance of 10.2 cm from the quartz window of a batch Xenon SteriPulse XL PUV sterilizer (Model RS 3000C) for 60, 120 and 180 s with
13 47.4, 94.8 and 144.2 J/cm 2 of energy respectively. It was observed that band intensities lactoglobulin and lactalbumin were decreasing in SDS PAGE for PUV treated whey protein. T he band for casein was not visible when the sample was treated for 60 s. Ne ither ban d was observed for either PUV treated whey or casein in Western blot. The indirect ELISA results showed a significant difference ( = 0.05) in IgE binding capacities between the PUV treated samples and control. Along with PUV, a sample size of 1.5 ml isola ted egg protein was treated in an Avure lab scale high pressure processor (model PT 1) at 600 MPa for 5, 15, and 30 min at three initial temperatures of 4, 21, and 70 o C Electrophoretic profiles by SDS PAGE showed that band intensities of all major egg al lergens were decreased after 60 s of PUV treatment and band intensities were not decreased after HHP treatments. Western blotting with pooled sera of allergenic patients did not show much decrease in band intensities for all major egg allergens with HHP tr eatments except at 21 o C Whereas, bands disappeared after 120 s f or PUV treatment, except for ovalbumin that exhibited a reduction in its band intensity HHP did not show any other decrease Indirect ELISA confirmed a significant increase and decrease ( = 0.05) in IgE binding of HHP and PUV treated samples respectively. Blueberry wine was treated with Xenon PUV sterilizer (model: RC 847) to understand the effect of processing on food safety and quality. Five gram of Red Star Montrachet culture ( Saccharomyc es cerevisiae ) was grown in 50 ml of Yeast Extract Peptone Dextrose ( YPD ) broth for 9 h at 30 o C and added to blueberry wine. Five ml (2.5 mm thickness) of inoculated blueberry wine was placed in an aluminum dish and subjected to PUV illumination for 12, 1 4, 16, 18, 20 and 22 s at a distance of 6 cm from
14 the quartz window of a Xenon PUV sterilizer (model: RC 847). Additionally, 10 ml (5 mm thickness) of the sample was treated for 25, 28, 31, 34, 37 and 40 s and 15 ml (7.6 mm thickness) of the sample was tre ated for 40, 44, 48, 52, 56 and 60 s at the sam e distance. A reduction o f 10.7 10. 7 and 9.6 log cfu/ml of Saccharomyces cerevisiae was observed at 22, 40 and 60 s treatments respectively. Five, 10 and 15 ml of samples treated for 12, 25 and 40 s resulted in 1.3 0. 8 and 0. 6 log cfu/ml reduction of Saccharomyces cerevisiae respectively. Ten ml of white grape wine was treated at the same distance gave 3.9 and 5.9 log cfu/ml reduction of Saccharomyces cerevisiae at 28 and 40 s treatment. There was no signifi cant difference in oxygen radical absorbance capacity (ORAC), total phenolic content, flavonoids, an thocyanins, lightness and hue at 30 s PUV treatment for treated and untreated blueberry wine samples (15 ml) GC MS analysis of PUV treated sample for volat ile aroma did not show the presence of new compound s compared to control. However, a significant difference was observed in ORAC, total phenolics, flavonoids and anthocyanins of thermally treated and untreated blueberry wine samples. Results indicated no m ajor detrimental effect of PUV on total phen olics and antioxidant activity for blueberry wine while inactivating microorganisms. Hence, PUV techno log y has great potential for the application of microbial inactivation and antigen mitigation with minimal eff ect on color, aroma, antioxidant activity and polyphenols depending on the food matrices. Key words: PUV, allergy, nonthermal, polyphenols, wine
15 CHAPTER 1 INTRODUCTION F ood processing and preservation techniques have been applied to ensure the safety o f food Though several techniques such as heat treatment, cold temperature treatment, irr adiation, microwave heating, pulsed electric field high pressure processing, and ohmic heating are available, there is always a n eed to investigate new technologies a s an alternative to existing processes to improve efficiency, reduce cost, improve yield with minimal quality changes. Pulsed ultraviolet light (PUV) processi ng is getting more attention as a better process for food safety (Krishnamurthy and Demirci 2006) Food allergies are important health problems in industrialized countries as nearly 2% of the adult population and 8% of children are affected by food allergies ( Monaci and others 2006) and the symptoms are exhibited among 22% of the general popul ation (W oods and others 2002). More than 160 food materials are identified as allergenic and eight of them including egg and milk lead to more than 90% of food allergies (Poms and others 2004). There have been several techno log ies utilized so far for mitigation of milk and egg allergens. They include heat, enzymatic, high pressure treatments, and food irradiation. However, loss of nutritional quality, development of off flavors, bitterness, expensive ingredients and the need for a specialized treatment system make it unsuitable for parti cle applications. PUV was proven to be effective in mitigating antigenicity of shrimp, almond, peanut and soy protein (Chung and others 2008 ; Yang and others 2011a ; Yang and others 2011b; Shriver and others 2011a) Hence, PUV has a p otential application in reducing the antigenicity of other major allergens such as milk and egg and this had become one of the focuses of this study.
16 The growth and metabolism of yeasts ( Saccharomyces cerevisiae ) and bacteria ( Oenococcus oeni ) results in the production of wine (Millet and Lonvaud Funel 2000) and wine may also contain acetic acid bacteria, other species of lactic acid bacteria and yeast which produces volatile acidity, off flavors and polysaccharides, and thus affects the wine quality. The undesirable microorganisms in wine include the yeast Brettanomyces sp p ., the bacteria Acetobacter aceti Pediococcus damnosus and several Lactobacillus sp p (Mi llet and Lonvaud Funel 2000). Ten to 20 pasteurization units (PU) are applied to beer for decont amination of spoilage microorganisms (King and others 1978). However, t hermal treatment has been proven to reduc e heat sensitive micro and macro nutrients and wines that are rich source s of polyphenols and antioxidants are believed to have similar detriment al effect on these micronutrients with thermal treatment. PUV is an emerging nonthermal techno log y that has been proven to be effective in inactivating microorgan isms in several food products. With higher antioxidant capacity, blueberries and blueberry pro ducts have gained more interest for the ir possible health benefits (Lee and others 2002). Several researchers investigated the antioxidant capacity of different Vaccinium sp p. but processing effe cts on compositional changes were less investigated. Justif ication of Study For children from birth to two years old, milk all ergy is the most common allergy and e gg is the second leading cause of food allergy in children. Avoiding milk and egg has been the only cure for children who are sensitive. Most of the we aning foods contain milk and egg as an ingredient. Inadvertent consumption of these foods can cause severe health problems to chi ldren who are sensitive to milk, t herefore it has become essential to remove allergens present in milk and egg Several process ing
17 techniques were applied to reduce or remove allergens in milk and egg Depending upon the sensitivity to treatment, few allergens were removed but not all major allergens. Thus the necessity for developing a new techno log y to reduce the a nti genicity of milk and egg without damaging its nutritional quality is needed PUV was shown to be effective against peanut soy wheat and shrimp allergens and therefore potentially may also mitigate milk and egg allergens also. Traditionally sulfit es or thermal paste urization have been used to make wine safe for consumption. Sulfites are chemi cal preservatives and thermal pasteurization degrades micro nutrients present in wine. With increase in demand for organic wine, a novel nonthermal techno logy that can inactivate microorganisms while improving the safety and quality but not affecting nutrients such as poly phenols would be a viable alternative. D epending upon the amino acid composition and molecular structure, proteins may exhibit different responses with UV radiat ion (Gennadios and others 1998) and PUV treatment This work will evaluate PUV treatment of three different food matrices, milk, egg and blueberry wine to evaluate antigen inactivation differences, microbial inactivation and quality as related to polypheno lics and antioxidant activity after treatment. Objectives The overall objective of this study was to understand the application of PUV for mitigating antigens and inactivat ing microorganisms in different food matrices. The specific objectives were to: F i rst, optimizing PUV protocol for removing allergens from isolated milk proteins and analyzing the effect of PUV by SDS PAGE, Western blot and Indirect ELISA
18 Second, o ptimiz ing PUV and HHP protocols for removing allergens from isolated egg proteins and ana lyz ing the effect using SDS PAGE, western blot and i ndirect ELISA. Third optimizing PUV protocol for inactivating Saccharomyces cerevisiae in southern high bush blueberry wine and white grape wine. F ourt h, analyzing the effect of PUV treatment on polyphen ols, antioxidant activity, color and aroma of southern high bush blueberry wine.
19 CHAPTER 2 LITERATURE REVIEW Nonthermal Food Processing Techno log y Consumers are expecting that food products should provide safety, variety, convenience, shelf life, nut ritional value, environmental soundness and reasonable cost. With low processing temperatures, low energy utilization, retention of flavors and nutrients, a fresh like taste, inactivating spoilage microorganisms and enzymes, nonthermal processes have been gaining importance in the food industry as an alternative for traditional thermal processing (Vega Mercado and others 1997) PUV techno log y has the ability to function at ambient or near ambient temperatures, and can avoid the deleterious effects that ther mal processing has on the flavor, color and nutrient values of foods (Krishnamurthy and others 2006) Pulsed UV Light UV light can be applied either in continuous mode or pulsed mode s In continuous mode, low, medium, or high pressure mercury lamps conti nuously emit radia tion in monochromatic or poly chromatic wavelengths with constant energy. Whereas in pulsed mode, capacitor stores the electric energy for a short period of time ( milliseconds) and releases it as ve ry short period pulses ( nanoseconds), whi ch transfers through a lamp (inert gas xenon or krypton) to ionize the gas and produces a broad spectrum of light (20,000 times more intense than that of sunlight) in the wavelength region of 100 nm (ultraviolet) to 1100 nm (near infrared). Spectru m of P UV light is divided into three regions as ultraviolet (UV) light (100 400 nm), visible light (400 700 nm) and infrared light (700 1100 nm). Vacuum UV (100 200 nm), UV C (200 280 nm), UV B (280 315 nm), and UV A (315 400nm) are the four sub regions of U V
20 light (Perchonok 2003). Though total energy generated is comparable to conventional UV light, PUV has several thousand times higher instantaneous energy which makes it four times more effective in pathogen inactivation ( Krishnamurthy and others 2009 ) P hotons are discrete fundamental pa ckets of energy for l ight. They have no mass, no electric charge and have an indefinitely long life span Photons contain a huge (Equation 2 1) hc/ (2 1) E = energy of photon, 34 J.s), c = speed of light in vacuum and Energy of the photon decreases with an increase in wavelength of light a nd hence, p hotons in the ultraviolet region have more energy than in the visible and infrared region. The ultraviolet region accounts for 54% of the total energy produced by PUV, followed by visible region (26%) and infrared region (20%) (Krishnamurthy and others 2006) UV light can contribute to break most of the chemical bonds such as covalent bonds in org anic compounds. Based on energy levels, UV, visible and infrared lights can ionize, vibrate and rotate molecules respectively. Molecules are elevated to an excited state upon absorbing energy. They can return to the ground state either by releasing the ene rgy as heat or photon. These excited molecules can even induce some
21 chemical changes. This can result in chemical and temperature changes within microorganisms and causes death (Krishnamurthy and others 2009) Variables The review of literature ( Chung and others 2008; Yang and others 2011a; Yang and others 2011b; Shriver and others 2011a ) reveal s the importance of sample thickness, treatment time, distance from UV source in PUV treatment. These are three critical variables to be considered during experiment al design. Other factors which can influence the efficiency of pulsed light are pulse width and pulse rate. A typical pulsed light operates at 1 20 pulses/s with pulse width of 300 ns 1 ms (K rishnamurthy and others 2009 ) Sample T hickness When light falls on a food surface, a portion of the light is absorbed by the food, while the rest is reflected, transmitted and scattered. As light penetrates (initial intensity I o ) through the food material (transparency coefficient T), its intensity (I) decay s along a distance of x from the food surface and is represented by Equation 2 2 (Palmieri and others 1999) I = T Io e x (2 2) Penetrated light decays exponentially throughout the sample. Intensity of this light drops below 37% after it reaches the penetration depth. Energy associ ated with molecules will be increased with the absorption of photons from the light. Photon count decreases with decrease in the intensity of light and less number of photons will be available for the molecule to absorb below penetration depth. Hence, with the increase in thickness of the sample, intensity of light decreases and results in less photon absorption and less energy association while penetrating through food. Thus,
22 penetration depth (x) of the sample plays a critical role in determining the thic kness of sample to be exposed with PUV. Less transparent foods have to be treated in a thin layer to overcome the penetration limitation. Wavelength of light has a significant effect on the absorption of light as absorption decreases with increase in wavel ength and results in low energy availability. Batch and continuous are the two modes in which pulsed light can be applied. In batch mode, sample s are kept in a stationary position and illuminated with PUV. In continuous mode, liquid sample will flow th ru a transparent tube and solid sample will be placed on a conveyor belt for PUV illumination. Diameter of the tube through which sample is flowing and flow rate of the sample plays a critical role in adjusting thickness of the liquid sample in continuous mod e. Krishnamurthy (2006) applied pulsed light for inactivating S taphylococcus aureus S uspension ( 12, 24, and 48 ml ) was transferred into an aluminum container with 7 cm diamet er for pulsed light treatment. Log cfu/ml reductions for S. aureus of 7.5, 4.6 a nd 1.5 were obtained for 12, 24 and 48 ml of sample respectively, when treated for 1 s (3 pulses) at 8 cm from quar tz window. When milk was treated for 105 s at the same distance, 2.13 + 0.28 and 0.16 + 0.07 log cfu/ml reductions of S. aureus were obtained for 12 and 48 ml of sample respectively. S. aureus inactivation was reduced with increase in sample volume as it increased thickness of sample. Illumination of 5.6 J/cm 2 PUV light (135 pulses) on 2 mm thick clover honey reduced 39.5% of Clostridium sporongene s, whereas no reduction was observed with 8 mm thick sample (Hillegas and Demirci 2003). Initial bacterial population in milk was reduced to 28% and 43% at a depth of 1 mm and 2 mm respectively with 4 pulses of 6 J/cm 2 PUV treatment.
23 Inactivating microorga nisms in liquids with high UV absorbance must be treated as a thin layer to make less UV available for liquids to absorb (Om s Oliu and others 2010). Treatment T ime Duration of pulsed light exposure to food is treatment time. In batch mode, sample will be kept at a constant position and is treated for a certain time. Light can be applied from any side. In continuous mode, treatment time is m easured in terms of flow rate for the sample. Higher flow rate, lowers the residence time (treatment time) for pulsed light illumination and results in lesser exposure of light. Shriver and others (2011 a ) reported a decrease in antigenicity of tropomyosin in shrimp extract with increase in treatment time (0 to 6 min) during pulsed light treatment at a distance of 10 cm from quar tz window. When blueberries were treated at 3 cm from quartz window for 5 s and 60 s, 1.3 + 0.6 and 4.9 + 2.4 log cfu/ml reductions of E. coli O157:H7 were observed respectively H owever there was no significant difference between 5 and 10 s treatmen ts ( Bialka and Demirci 2007) As the treatment time increases, sample has more exposure to pulsed li ght and accumulates more energy b ut prolonged treatment times can lead to elevated temperatures that might adversely affect the food quality According to W ang and others (2005), Xenon flash lamp treatment at 254 nm for inactivation of E. coli is no different from continuous UV low pressure mercury lamps at the same wavelength; however, PUV has an advantage of rapid disinfection. PUV treatment at 270 nm showe d maximum reduction while treatments above 300nm did not inactivate E.coli Distance from Light S ource The intensity of PUV is proportional to the distance from its source. Sample placed near to the central axis receives the highest energy. Maximu m energy that can
24 be absorbed by the sample decreased with increase in distance of the sample. Surface of the sample can receive maximum energy of 0.37, 0.33, and 0.22 J/cm 2 energy at a distance of 9.6, 13.2 and 17.0 cm from the light source of a Steripuls e XL 3000 manufactured by the Xenon Corporation (Woburn, MA) respectively (Krishnamurthy 2006). When blueberries were treated for 60 s at a distance of 3 and 13 cm from the quartz window, a 4.9 + 2.4 and 3.0 + 0.6 log cfu/ml reductions of E. coli O157:H7 res pectively were observed, however there was no significant difference between 3 and 8 cm treatments. The UV dose received by the sample at 3, 8 and 13 cm after 60 s treatment time was 32.4, 22.6 and 12.4 J/cm 2 respectively ( Bialka and Demirci 2007) Deconta mination of food products was decreased when placed very close to the lamp as the position and orientation of the lights can also affect the incident energy on the product (Oms Oliu and others 2010). Inactivation efficiency of PUV is higher in a sample pla ced near to the central axis of the lamp. Factors Affecting PUV E fficacy F ood product s are composed of different components and each component has different properties for absorption of light, and this makes composition as a critical factor in PUV efficie ncy. Fruits and vegetables (high in carbohydrates) are better absorbers of UV light than proteinaceous or oily foods for inactivating L isteria monocytogenes P hotobacterium phosphoreum and C andida lambica using PUV. Part of PUV can be absorbed by proteins or oil, which reduces the efficiency of the treatment (Oms Oliu and others 2010). The nucleic acids p urine and pyrimidine bases absorb strongly in the near UV region Amino acids such as tyrosine, phenylalanine and tryptophan absorb strongly in
25 UV region. Hence, food samples containing these compounds absorb more UV light than other food samples (Koutchma and others 2010). Vegetal tissues containing chlorophyll, carotene and xanthophyll and meat tissues containing myoglobin, melanin, hemoglobin, deoxyhemogl obin, cytochrome C oxidase absorb more visible light. Water absorbs most of the near infrared light as it has an absorption peak at 970 nm. Fat and casein also absorbs near infrared light. Shielding effect or shadow effect can minimize the efficacy of PUV on a food product with high counts of microorganisms. Microorganisms present in upper layer will be inactivated with PUV illumination but they also provide shadow ing to the rest (microorganism) from light and mak e this treatment less effective. Crevices, injuries or irregularities on food surfaces can hide microorganisms from light exposure and thus protecting them from inactivat ion (Oms Oliu and others 20 10). PUV has higher efficiency i n water compared to other liquids such as sugar solutions and wines a s solids present in these solutions minimizes the penetration of PUV Clarified fresh juices and high pulp juices vary significantly in absorbing UV An absorption coefficient of clarified apple juice is 11 per cm and orange juice is near 50 per cm. The pr operty of absorbing more light by dark products than light products resulted in higher color change for black pepper compared to wheat flour (Fine and Gervais 2004). Bialka and Demirci (2007) reduced 4.3 and 2.9 log cfu/ml of Salmonella and E. coli O157:H7 respectively in blueberries with 22.6 J cm 2 of energy (60 s treatment). Anderson and others ( 2000 ), attributed resistance of Aspergillus niger spores to the protective dark pigment present in cell wall layers which absorb s strongly in the UV range. Tuto r i and Nicolau (2007) suggested a similar mechanism of absorbing more
26 light energy by pigment for higher inactiva tion of dark colored phialo s p ores produced by A. niger (black) and A. cinnamomeus (brown) compared to A repens (green). Moeller and others (20 05) studied the effects of UV radiation on spores of red pigmented B atrophaeus DSM 675, dark gray pigmented B atrophaeus T DSM7264 and light gray B subtilis DSM 5611. All three strains responded identically to UV B and UV C radiation, where as dark red pigmented spores were 10 times more resistant to UV A illumination compared to the other two spores. The r UV limited against U V A, but not against UV B and UV C regions. Several researchers (Krishnamurthy and others 2006; Oms Oliu and others 2010) concluded UV germicidal effect as a major mechanism for inactivating microorg anisms during PUV treatment and pigments that cannot absorb UV C light have minimal microbial inactivation Surface topography of product or package has a complex role in inactivating surface microorganisms using PUV. H igh hydrophobic and reflective nature of surfaces with smooth finish es reduces the effect of PUV on surface microbial inactivation (Woodling and Moraru 2005). Energy of 1.25 J/cm 2 inactivated S. aureus on the surface of various packaging materials inoculated with 10 1000 cfu/cm 2 and energy of >2 J/cm 2 inactivated B cereus and Aspergillus spp. spores. Paper polyethylene packaging materials inocul ated with molds such as A niger A repens A cinnamomeus and C. herbarum were significantly decontaminated using PUV treatment (Tutori and Nicolau 2007). Rough surfaces provide a shield for microorga nisms from PUV illumination and hence reduce the efficiency of inactivation. Opaque nature of cereals, grains and spices
27 makes them unsuitable for PUV treatment. It can be used as a final step during minimal processing for deconta mination or sterilization of a product. In order to achieve the germicidal advantage of PUV, the sample has to be positioned in a way that cannot avoid photons and microorganisms contact. Any obstruction can restrict the efficiency of PUV treatment. To achieve r equired surface deco ntamination utilizing PUV treatment precautions such as ensuring the exposing of entire surface to light, selecting food products with minimal irregularities and minimizing food p roduct rolling over each other ha s to be followed For liquid samples, optim ization of flow rate, considering absorption coefficient, the amount of solids and thickness of the sample during PUV process design can yield better results. Decontamination efficiency decreases with increase in microbial density since microorganism s fou nd in the top layer receive photons for inactivation while those i n the bottom layer experience a shadow effect. Virus inactivation was reduced in protein supplemented buffered saline solution compared to regular buffered saline solution with PUV treatment (Roberts and Hope and 2003). PUV treatment reduced >7 log cfu/ml K lebsiella and >4 log cfu/ml poliovirus, rotavirus and Cryptosporidium parvum in water (Huffman and others 2000). Photosensitizers are used to improve the anti microbial action by providing a synergistic effect. The s ynergistic effect of reactive molecules and radicals ge nerated by photosensitizers in the sample increases the efficiency of PUV treatment. Effectiveness of PUV treatment is dependent of the composition of light spectrum emitted from the source (Oms oliu and others 2010). The absence of UV C in PUV spectrum resulted in reducing the inactivation of A.niger spores on polyethylen e terephthalate (PET) surface by three to five fold (Wekhof and others 2001). High and low content of UV i n the light
28 spectrum resulted in inactivation of 6 and 2 log cfu/ml respectively for E coli L monocytogenes, S enteritidis, P aeruginosa, B cereus, and S aureus with 200 pulses (Rowan and others 1999). Pulsed UV L ight Applications Several researcher s reported PUV efficacy in inactivating microorganisms in several food products, reducing antigenicity of isolated proteins and decontaminating surfaces. PUV treatment also increased Vitamin D 2 content in white button mushroom to 395 g/g DM when treated f or 10 s at a distance of 15.8 cm from UV source (Marry 2009) Ina ctivation of M icroorganisms Several studies have confirmed the inactivation of bacteria, yeast, mold and viruses. Effectiveness of PUV varies with the state of a food product. Its function v aries from sterilization of a liquid product depending on its absorption coefficient and degree of transparency to surface inactivation of a solid food product or packaging material. Wekhof and others (2001) reported 4.8 log cfu/ml reductions for A. niger spores treated with 5 PUV flashes at 1 J/cm 2 Lamont and others (2007) reported 4 and 1 log cfu/ml reduction of poliovirus and adenovirus with 10 pulses at a dose of 12 mJ/cm 2 respectively. Application of PUV is more suitable for the surface sterilization than compared to its utility for liquid sterilization. However, minimizing sample depth, increasing treatment time and reducing distance of the sample from light source can improve the inactivation of microorganisms in liquid medium. Results were reproduci ble and independent of initial load in inactivating microorganisms in clear liquids (Uesugi and other 2007). Microbial inactivation in clear liquids can be predicted using W eibul model, however it fails for products with substrate interaction (Uesugi and o ther 2007)
29 The mechanism for inactivating microorganisms with PUV treatment is attributed mainly to photochemical, photothermal and photophysical effects. However, photodynamic effects play a vital role in in activating microorganisms in sample s with photo sensitizers. PUV can induce photodynamic effect in samples with photosensitizers such as riboflavin enhanc ing its efficacy. Photothermal effect can be observed in samples wh en the temperature is increased with the absorption of higher amount of energy. Th is heat energy has a synergistic effect with PUV in inactivating microorganisms. The difference in absorption of UV light by bacteria and surround ing media results in overheating within bacteria and causes bacterial disruption by photothermal mechanism s wi th the exposure of >0.5 J/cm 2 en ergy (Elmnasser and others 2007; 2008). Treating minimally processed vegetables such as spinach, radicchio, lettuce, cabbage, carrot, green bell pepper and soybean sprouts for 180s/side at a distance of 12.8 cm from light st robe reduced 0.56 to 2.04 log cfu/g mesophilic aerobic microorganisms (Gomez Lopez and others 2007). However, increase in treatment times can generate high heat in the sample that is undesirable for most of the PUV applications for food and makes this a ma jor limiting factor. Photochemical e ffect UV targets the DNA molecule in microorganisms and hence this nucleic acid becomes the primary target for PUV. Photochemical tran sformation of pyrimidine bases form s dimmers in the DNA of bacteria, viruses and ot her pathogens and this was the source of the germicidal activity of UV light. It prevents DNA unzipping, inhibits replication and results in mutation, impaired replication and gene transcription in the absence of repair mechanisms, which leads to death of the organism. Mechanisms that are responsible for damaging DNA during UV treatment are reversible under certain
30 conditions (wavelengths >330 nm). Protein factors can repair the DNA damage. Activated photolyase enzyme monomerises dimer species and photoreac tivates split nucleic acid (Guerrero Beltran and Barbosa Canovas 2004). However, PUV treatment can inactivate DNA repair system s and researchers have shown that PUV treated organisms do not enzymatic ally repair DNA. Damaged DNA, s ingle strand breaks and p yrimidine dimmers were formed in yeast cells during PUV treatment. It was observed that UV induced more DNA damage in yeast cells compared to PUV, however inactivation of yeast cells remained the sa me, suggesting the contribution of mechanisms other than D NA damage possible some photochemical mechanism during PUV treatment. Formation of pyrimidine dimmers including thymine dimmers during UV light processing causes germicidal effect of PUV treatment. Dimers cause clonogenic death (inability to replicate) by inhibiting the formation of new DNA chains during cell replication DNA absorbs UV during PUV treatment of bacterial spores and results in photochemical effect with the formation of s pore photoproduct 5 thyminyl 5,6 dihydrothymine (Gomez Lopez and others 2005 b ). Photo t hermal e ffect When the muscle side of a salmon fillet was subjected to PUV treatment at a distance of 8 cm from UV strobe for 60 s, a maximum reduction of 1.09 log cfu/ml E. coli O157:H7 and 0.74 log cfu/ml L monocytogenes was observed. A t reatment at 5 cm distance f or 30 s on the skin side of salmon fillet reduced 0.86 log cfu/ml of E. coli O157:H7 and at 8 cm distance for 60 s reduced 1.02 log cfu/ml of L monocytogenes Skin side treated with PUV had higher surface temperatures compared t o muscle side and this additional heat energy may be contributing to higher microbial inactivation on the skin side (Ozer and Demirci 2006). Compared to continuous UV treatment, PUV
31 treatment caused more DNA damage in S cerevisiae cells as increase in fl uence caused more protein elution from yeast cells after the treatment (Takeshita and others 2003). Vacuole expansion and cell membrane distortion in yeast cells treated with 1.4 J/cm 2 of PUV treatment was also reported by Takeshita and others (20 03). Acco rding to Fine and Gervais 2004 a p rolonged treatment result s in overheating and cause s vaporization that generat es a small steam and destr oy s membrane s Structural changes in DNA disintegration of cell s due to instantaneous overheating of cell constituen ts and UV induced thermal stress to rupture bacteria results in microbial inactivation (Wekhof and others 200 1 ). Krishnamurthy and others (2008) observed cell wall damage, cytoplasmic membrane shrinkage, cellular content leakage and mesosome disintegration in S aureus with transmission electron microscopy and Fourier transform infrared spectroscopy at 5 s P UV treatment. Electron micrographs of PUV treated A niger confirmed the rupture and/or deformation of the spore s These results provide evidence about the loss of overheated content after internal explosion and evacuation during PUV treatment (Gomez Lopez and others 2007). Takeshita and others (2003) observed the effect of continuous wave UV light (CW UV) and PUV on S cerevisiae and concluded that simi lar DNA damage and subsequent formation of single strand breaks and pyrimidine dimmers in yeast cells occurred Cell membrane damage was evident with increased concentration of eluted protein and structural changes in PUV treated cells. PUV treatment expan ded vacuoles which distorted/damaged cell membrane s and changed their shape to circular, while CW UV did not alter the yeast cell structure. Even before reaching 58 J/cm 2 for the decontamination threshold in food powders, their visual and flavors qualities were
32 affected (Fine and Gervais 2004). Thermal mechanism dominated the UV effect for colored food powders and overheating, oxidation caused color changes in black pepper and wheat flour prior to the decontamination threshold. Photophysical e ffect Spores a nd vegetative cells respond differently to UV due to their structural differences. Thymine dimers are formed in vegetative cells during UV exposure are not observed in detectable levels in bacillus spores (Krishnamurthy and others 2009) 5 thyminyl 5,6 dih ydrothymine adduct is a photo product formed in spores during UV radiation. K rishnamurthy and others ( 2009) hypothesized that higher flux densities lead to thermal stress on bacterial cells causing cell rupture. Different heating and cooling rate of bacter ia and surrounding medium during PUV treatment induces localized heating. Intermittent high energy pulses cause structural damage to bacterial cells and leads to the inactivat ion of microorganisms Photod ynamic e ffect Photosensitizers can be used to improv e the anti microbial action by providing a synergistic effect. Photosensitizers generate reactive molecules and radicals from the sample and results in t he synergistic effect with PUV that increases the efficiency of treatment. McDonald and others (2000) us ed hydrogen peroxide (H 2 O 2 ) as a photosensitizer, and observed that a PUV treatment (fluence of 12 mJ/cm 2 ) with H 2 O 2 w as more effective as it inactivated 2 log cfu/ml more B subtilis spores in aqueous suspension compared to PUV treatment alone. UV light can produce oxygen radicals at 185 to 195 nm during processing which can form ozone and cause off flavors in food (Krishnamurthy 2006). Riboflavin is a strong photo sensitizer that can absorb visible and UV light and transfers this energy into highly react ive singlet oxygen. This singlet
33 oxygen can lead to several oxidation reactions in milk. Oxidation of methionine produces dimethyl disulfide and secondary lipid oxidation products such as hexanal and pentanal that are major light induced off flavors in mil k (Mestdagh and others 2005). Natural food grade photosensitizers can be used to decontaminate food and increase its shelflife H aematoporphyrin, sodium chlorophyllate, riboflavin, hypericin, and psoralen are plant food con stituents which can be used as p hotosensitizer s (Kreitner and other 2001 ; Luksiene 2009). Kreitner and others (2001) introduced a new combination of visible light and photosensitizers. Researchers inactiva ted 3 to 5 log cfu/ml g ram positive bacteria and 0.3 to 3 log cfu/ml yeasts by using haematoporphyrin and sodium chlorophyllin as photosensitizers in a photodynamic treatment. Photosensitization with 5 aminolevulinic acid significantly inactivated vegetati ve cells and spores of B cereus in vi tro (Luksiene and others 2009). Irradiation with visible light can produce bio log ically reactive oxygen species such as singlet oxygen and superoxide radicals that are highly effective in inactivating microorganisms an d free radicals that causes cell disintegration. Light energy transferred to tissue and cells by photosensitizers, generates reactive cytotoxic species. Membrane components are highly sensitive to photodynamic treatment. Application of photosensitizers dur ing PUV treatment can lower the treatment time while achieving the similar microbial reduction It also helps in avoid ing heat generation that adversely affects the sensory properties. However, possible off flavor (such as lipid oxidation) generation shoul d be considered while selecting the sample. A photosensitizer used in a food should have food additive status, low cost, high chemical purity and be effective at low concentrations. It should
34 not produce mutagenicity or genotoxicity, imp art color, flavor a nd taste, nor a ffect nutritional and organoleptic properties of foods (Luksiene 2009). Melanoidin, a final product from nonenzymatic browning or Maillard reaction acts as a photosensitizer to UV A radiation and produces singlet oxygen (Argirova 2005). Phot oreactivation One of the main concerns for light treated sample s is getting recontaminated by photoreactivation. Illuminating ultraviolet treated bacteria with visible light can reverse the induced ultraviolet damage by photoreactivation. Photolyase is an enzyme (flavoprotein) catalyst that splits UV induced cyclo butane dimmers in damaged DNA using light energy through a radical mechanism. It contains two noncovalently bound chromophores in which one chromophore is a fully reduced flavin adenine dinucleoti de (FADH ) and the other is methenyltetrahydrofolate or deazaflavin. Deazaflavin harvests sunlight and increases the repair efficiency by resonating energy to FADH which otherwise directly absorb s photon s to get excited for executing repair function. An e lectron is then transferred to pyrimidine dimmer from excited flavin cofactor to produce a charge separated radical pair. The d excess electrons to flavin radicals (to restore catalytic form of FADH ) and closing the catalytic photocycle (Gomez Lopez and others 2007). Otaki and others (2003) observed photoreactivation after PUV treatment, however the rate at which it progresses is slower compared to the rate after CW UV tre atment. Researchers used alumin um foil to wrap their samples to evade photoreactivation. Gomez Lopez and others (2005) reported photoreactivation in PUV treated cells. Other possible repair mechanisms for UV damaged cells are dark repair mechanism that occurs in the absence of light and s pore photoproduct repair mechanism or common
35 excision repair mechanism through which spores can repair themselves. Illuminating with longer wavelengths such as visible light can reverse photo chemical effects and result in photoreactivation. The r ate of pho toreactivation was higher after continuous UV light treatment compared to PUV. Variation in protective and repair mechanisms against the damage and difference s in cell wall composition of bacteria may be responsible for different responses from microorgani sms. However, m ore research has to be conducted before confirming photoreactivation phenomenon after PUV treatment. Effect of PUV on Food Q uality PUV treatment on quality and sensory properties of food products has not been comprehensively studied. PUV off er s an advantage of limited oxidative reactions during treatment with its lower pulse duration (300 ns to 1 ms) compared to continuous UV light. Milk treated with PUV did not have any significant changes in amino acid composition and lipid oxidation (Elmna sser and others 2008). Potato slices that were treated with 3 J/cm 2 retained color after prolonged storage compared to untreated sample s suggesting possible inhibition of browning (Dunn and others 1989). Higher enzy me activity was observed for polyphenol o xidase extracted from control compared to PUV treated sample. Human health enhancing chemical substances such as scoparone in grapes, 6 methoxymellein in carrots, resveratrol in grapes and anthocyanins in strawberries and apples can be induced using UV tre atment. PUV treatment (30 J/cm 2 ) did not affect protein, nitrosami ne, benzopyrene and vitamin C in frankfurters compared to untreated samples According to Dunn and others (1995), riboflavin content in beef, chicken and fish were not reduced using PUV. P ul ses (2 4) of 2.5 5 J/cm 2 of PUV treatment did not change hunter color and shear forces in pre packaged catfish fillets (Shuwaish and others 2000). Salmon treated with PUV at 3 cm
36 distance for 30 s and 5 cm distance for 45 s was overheated resulting in visu al color changes for the sample (Ozer and Demirci 2006). White cabbage, iceberg lettuce, leek, paprika, carrots and kale were treated with three pulses of PUV and stored at 7 and 20 o C for up to 7 days to observe any change in sensory properties. PUV tre atment and storage did not affect sensory properties for all vegetables except iceberg lettuce that suffered discoloration. Minimally processed white cabbage was treated for 45 s/side at a distance of 12.8 cm from strobe and stored at 7 o C for 9 days to ob serve its sensory properties such as off odor, taste, overall visual quality, sogginess, browning and dryness. Panelists observed off odor (plastic) in the sample which was presented immediately after treatment; however the off odor was disappeared after f ew hours. The tested sensory parameters were rated below the rejection limit by panelists till 9 days, except for off odor which was unacceptable after 7 days. PUV treated iceberg lettuce was acceptable (score 3) for taste, off odor, sogginess, leaf edge b rowning, leaf surface browning, translu cency and wilting after 3 days storage at 7 o C, however, it scored par below acceptable limit for overall visual quality (OVQ). Although the contribution of browning for OVQ had not been determined, leaf edge browning and leaf surface browning were considered as the reason for low OVQ. They concluded that PUV might not have inactivated polyphenol oxidase enzyme that caused browning in iceberg lettuce (Gomez Lopez and others 2005). PUV treatment did not affect the funga l growth on strawberries and did not reduce the sepal quality decay rate during storage at 12 o C for 10 days (Lammertyn and other 2003). There was an i ncrease in respiration rate of iceberg lettuce by 80% and was not observed in a similar treatment of whit e cabbage (Gomez Lopez and others 2007).
37 FDA considers the change in chemical composition of food including the influence of the treatment on nutrients while assessing the safety of foods treated with radiation and it approved PUV treatment under the code 21CFR179.41 (Dunn and others 1997 and Gomez Lopez and others 2007). Formation of toxic by products in any food sample treated with PUV is not reported and based on the nature of light used in PUV, it is assumed that no radioactive by products would be pro duced. PUV has a potential to replace chemicals (which cause eco log ical problems) for disinfection purposes. The absence of mercury in Xenon PUV lamps make them environmental friendly compared to CW UV lamps. However, l onger treatments result in heat accum ulation which causes undesirable visual and flavor changes to the product and hence PUV can be used as a rapid disinfectant for surfaces and clear liquids.
38 CHAPTER 3 EFF ICACY OF PULSED ULTRAVIOLET LIGHT ON ANTIGENICITY OF ISOLATED MILK PROTEINS Backgro und Food allergies are abnormal immuno log ical reactions to a food or food component (Poms and others 2004). These are important health problems especially in industrialized countries, since 1 2% of the adult population and 8% of children are affected by fo od allergies ( Monaci and others 2006), and yet certain symptoms of food allergy are exhibited among 22% of the general population (Woods and others 2002). Food allergens can be defined as those substances in foods that initiate and/or provoke the immuno lo g ical reactions of allergy (Poms and others 2004). More than 160 food materials are identified as allergenic, but eight of them including milk lead to more than 90% of food allergies (Poms and others 2004). According to Directive 2003/89/EC, there are twe lve allergenic ingredients whose presence has to be declared and milk is one of them (Monaci and others 2006). In children from birth to two years old, milk allergy is the most common allergy. A pproximately 1.6 to 2.8% of children less than two years of ag e have exhibited symptoms for cow milk allergy (Poms and others 2004), b ut, after the age of three, 85% of these children will outgrow their allergy. According to various studies based on populations in different countries, 0.3 to 7.5% of the total populat ion is affected by cow milk allergy (EI Agamy 2007). Avoiding milk has been the only cure for children who are sensitive. However, most weaning foods contain milk as an ingredient. Also, milk and its derivatives have been used in a wide variety of foods, c ausing increase in unexpected consumption. Inadvertent consumption of these foods can cause severe health
39 problems to children or adults who are sensitive to milk. Therefore, it has become essential to reduce antigenicity of milk. Milk protein consists of 80% casein and 20% whey, while the latter mainly consists lactoglobulin (4%) in total milk protein. Natale and others (2004) reported that the most allergic proteins in milk were caseins. Children can also show hypersensitivity lactoglobulin. Wal (2004) suggested that most proteins in milk are allergens despite their lower concentrations. Lee (1992) reduced the antigenicity of whey protein with heat treatment considerably. The enzymatic hydrolysis reduced t he allergenicity and antigenicity of protein. But enzyme hydrolysis can produce bitter peptides and off flavors which were attributed to liberation of peptides and amino acids from proteolysis. Several processing techniques were applied to reduce allergen icity of milk. But, depending upon the sensitivity to treatment, allergenicity associated with few major proteins were reduced but not for all major allergens. Thus, the necessity for developing a new techno log y to reduce the antigenicity of milk has evolv ed, and PUV is a potential technique, since several researchers have successfully used PUV to reduce food allergens and it was expected that PUV would be equally effective in reducing milk allergens. PUV is an emerging non thermal techno log y that consists of intense flashes of broadband lights under the wavelength from 100 to 1100 nm, with approximately 54% ultraviolet, 26% visible and 20% infrared spectra. The instantaneous energy of PUV can be thousands of times higher than that of the conventional UV li ght due to its short pulse duration in a few nanoseconds to microseconds, and each pulse can reach 90000 times
40 the intensity of sunlight at sea level. PUV is known for its superior capacity for surface disinfection such as for strawb erries and raspberries (Bialka and Demirci, 2008), pathogen inactivation ( Krishnamurthy and others 2004, 2005, 2008), enhancement of bioactive antioxidants and phytochemical contents in fruits and vegetable such as Napoleon table grapes where the resveratrol content was found to increase by 11 folds (Alothmana and others 2009), and water sanitation ( Auchincloss 2002 ). Most recently, Yang and others (20 11a ) and Chung and others (2008) have shown PUV can effectively reduce allergen potency of peanut butter. They reported that the a llergens Ara h 1 and Ara h 3 were significantly reduced by PUV treatments. The objective of this study was to explore the capacity of PUV in reduction or removal of milk allergens Materials and M ethods Equipment Steripulse XL 3000 manufactured by the Xen on Corporation (Woburn, MA) was used for PUV treatment. Electrophoresis gel equipment (Criterion TM cell serial no. 135BR 0015539) was used to conduct all SDS PAGE analysis with pre cast gels by Bio rad. U Quant micro plate reader, MQX200 by Bio Tek Instru ments Inc., (Winooski, VT) was used to quantify reduction of IgE binding by ELISA Experimental D esign The major objective of this study was to reduce the antigenicity of milk proteins using PUV. Energy studies reveal the importance of treatment time and d istance from the UV source to understand the amount energy available for a food product (Krishnamurthy 2006). Amino acid composition and molecular structure of proteins may cause different responses with UV radiation for different proteins (Gennadios and o thers
41 treatment time in PUV treatment. This was the critical factor considered in this study. A n experimental design with full factorial and three replications was us ed. The treatment conditions were set based on preliminary data ca sein were used in the study. T here w as a total of 15 treatments (3 x 5) conducted after the preliminary study with PUV for each sample. Materials The assay serum from a p ool of human sera of patients (Plasma Lab International, Everette, WA) with a documented history of milk allergy was used. Highly purified isolated milk protein extracts were purchased from Sigma Chemical Co. (St Louis MO). Isolated ACA (lyophilized powder Chromatographically purified, approximately 85% by electrophoresis) and whey extracts were used. C asein protein was dissolved in sodium p hosphate buffered saline (PBS) with NaCl of 0.15 M having pH of 7.4 and molarity of 0.01 M. The final concentration o f casein solution was adjusted to 2.0 mg/ ml Whey protein was also dissolved in the same buffer and adjusted to concentration of 40 mg/ ml Four ml of sample was treated at a distance of 9.6 cm from the UV source. The solution was transparent enough to get penetration of PUV light. Treatment times used for casein solution were 60, 90, 120, 150, and 180 s while for whey protein ; they were 60, 90, 120, and 150 s at second level. Allergen a nalysis Gel electrophoresis and western b lot Electrophoresis was used to compare and contrast the differences in protein bands for control and treated samples. Sample ( 37.5 l ) was added to 12.5 l of XT buffer 4X, followed by a heat treated for 10 min at 100 o C Ten g of protein was then
42 loaded in a gel (4 20% of Tris HCl) a nd ru n at 200 V for 1 h The gel was stained (Gelcode TM Blue safe protein stain solution Pierce) and des tained (distilled water) for 1 and 1.5 h respectively G el code staining solution was added for 1 h with shaking. Stain was removed using destainer fo r 1.5 h. Image was captured to observe the effects of PUV on milk proteins. Similar protocol was followed for running a gel for western blot and the electrophoresis was followed with placing a gel for 10 min in a transfer buffer. Trans Blot SD cell was u sed to transfer to an Immobilon P membrane by running at 10 V for 30 min and then blocked by immersing in a blocking buffer for 2 h at room temperature. Transfer membrane was washed with TBS containing 0.05% Tween 20 and this was continued after each step. Blocked tra nsfer membrane was incubated overnight with 1:5 ratio of primary antibody, and then incubated for 2 h in a 1:250 ratio of secondary antibody. One step chlronaphthol was added to observe IgE binding. Indirect ELISA Indirect ELISA was conducted to quantify the binding ability of IgE with milk ml ) was applied to the plate and then incubated for 2 h at 37 o C The plate was washed with tris buffered saline containing 0.05% Tween 20 (TBST) for three times a nd was repeated after each step. Superblock ( ) was applied to each well and incubated for 2 h at room temperature for and incubated for 2 h, followed with an incu bation of 1:1000 ratio horseradish peroxidase conjugated rabbit anti of substrate prepared using 5 mg o phenylenediamine dihydro chloride (OPD) in 9 ml of distilled water and 1 ml of stable peroxide buffe r, was added to each well for a color
43 2 SO 4 with change in color from clear to golden yellow. The absorbance was measured with the Spectra Max at 490 nm. Data A nalysis In the analysis of st atistical significance, ELISA reading was set as the dependent variable and distance from the central axis of the lamp and time as independent variables. The two protein samples were used. Tukey test was conducted for sample comparison at different levels of distance and time with the significant level of Results and D iscussion SDS PAGE and W estern B casein Krishnamurthy (2006) measured maximum energy available per pulse at different distances for a similar PUV light system. It is expected that a treatment of 60s, 120s and 180s at a distan ce of 10.2 cm from quartz window will receive 47.4, 94.8 and 144.2 J/cm 2 of energy. However, the actual energy absorbed by the sample depends upon several factors such as transparency, optical properties and presence of solids etc. PUV treated samples were analyzed for their electrophoretic profiles and IgE binding using SDS PAGE (Fig ure 3 2 ) and Western blot (Fig ure 3 4 ), respectively. A significant reduction in volume of the sample was observed when samples were treated with PUV. A 40% reduction was also reported by Chung and others (2008) after treating 10 ml of peanut sample with PUV. The reduction of volume was increasing with an increase in treatment time. The sample treated for 180 s had a higher moisture loss compared to 60s treatment. This moisture loss may be due to a rise in temperature that in turn gives enough kinetic energy for the water molecule to escape.
44 Figure 3 2 casein profile from SDS PAGE. During electrophoresis, marker was loaded in fir st lane followed by control, 60, 120 and 180 s treatments, s2 proteins are the major antigens present in casein and they were identified in the control. However, the intensity of these protein bands was o bserved to be decreased at 60 120 and 180 s treatment. Samples were loaded in a similar way for Western blot and r esults were reported in Figure 3 4 The bands which s1 s2 casein prot eins were identified in Figure 3 4 whereas no such band was app arent for treated samples. This indicates the absence of IgE binding in treated samples and possibly antigenicity associated with these proteins. Similar results were obser ved in SDS PAGE and western blot of PUV treated almond, peanut, A tlantic white shrimp and soybean protein extracts. Photochemical, photophysical and photothermal are three major mechanisms through which PUV inactivates microorganisms. Howev er, there is no confirmed mechanism for mitigating antigenicity with PUV treatment. PUV treatment can cause protein modification such as protein fragmentation, denaturation or crosslinking and can affect their binding ability with IgE (S hriver and others 2011 a ). P rote in fragmentation makes the protein migrate faster through the acrylamide po res leaving the gel and entering the chamber buffer. Protein crosslinking may make the protein large enough in size that it can not penetrate through the pores or it may reduce its velocity causing smearing in SDS PAGE and western blot. According to Cho and Song (2000) and Lee and Song (2002), irradiated protein can be damaged by fragmentation and aggregation. Formation of disulfide bonds, hydrophobic and
45 electrostatic interactions and generation of inter protein cross linking reactions can convert protein into higher molecular weight aggregates (Davies and Delsignore 1987). casein allergenicity was reduced after treating the protein with gam ma irradiation (Lee a nd others 200 1; Lee and others 200 5 ). The decrease in intensity of the band in SDS PAGE was the result of a slight breakdown in the polypeptide chain with the irradiation at low doses (Lee and others 2005). They observed cross linked products of degraded proteins which cannot penetrate through the running gel at high energy dose. SDS PAGE and W estern B lot for W hey P rotein Figure 3 1 and Figure 3 3 shows the analysis of PUV treated whey protein for molecular characteristics using SDS PAGE and IgE binding u sing Western blot, respectively. There was also a significant moisture l oss in the whey protein sample. There are two major bands obse rved for control in the Figure 3 1 and Figure 3 3 The band at 18 kDa is lactoglobulin and the other b and at 14 kDa is lactalbumin T he band intensities decreased with an increase in treatment time. A new band at 10 kDa was appeared in SDS intensity was found to be increased with increase in treatment time. The appearance of a new band suggests the possible fragmentation of whey protein into smaller proteins. Water in the sample can absorb UV photons and produce OH and H + radicals. Davies and others (1986) report ed that the hydroxyl radicals induce gross struc tural modification on proteins. These modified proteins can undergo spontaneous fragmentation or can exhibit substantial increases in proteolytic susceptibility. They demonstrated that any aspect of protein p rimary structure can be modified by OH They observed OH+O 2 +O 2 cause s spontaneous BSA fragmentation and produce new
46 carbonyl group s with no apparent increase in free amino groups. The formed amino acid radical in the peptide chain can cross link with an amino acid radical in another protein. Elmnasser and others (2008) observed the lactoglobulin after 5s treatment using pulsed light at 280 nm. There were no significant changes in the amino acid composition after this treatment. Cho and song (2000) observed the disruption of ordered structure of protein molec ules degradation, cross linking and aggregation of the lactoglobulin when they were gamma irradiated. Conformational change s w ere also observed in them. Milk proteins have aromatic amino acids such as phenylalnine and tyro sine, and the disulfide engaging amino acid cysteine. It is understood that nucleic acids are the strongest absorbers of 253.7 nm light. While purine and pyrimidine bases on the nucleic acid strands absorb 253.7 nm light, the polymeric backbone does not. C ompounds containing conjugated bonds (aromatic ring or double ring molecules) and disulfide bonds are effective absorbers at this wavelength, and confirm the absorption of UV light by milk proteins (Krishnamurthy and others 2010) Three essential amino aci ds histidine, phenylalanine and tryptophan can be degraded by UV light. The complex reactions during photo degradation of proteins lead to change s in solubility, sensitivity to heat, mechanical properties, digestion by proteases and physical properties. Up on absorption, 253.7 nm light can theoretically affect O H, C C, C H, C N, H N and S S bonds and causes direct photo degradation. Heat treatment over 60 o C sheets, barrel, and exposure of disulfide bonds and free cysteine. When heat ed
47 above 65 o C, protein denatures and forms irreversible aggregation with new hydrophobic interactions and disulfi de bond exchanges. Heat treatment at 80 to 90 o C increased antigenicity of BLG whereas the treatment above 100 o S1 casein S2 casein have 214 and 222 amino acids (Fi gure 3 6, 3 7, 3 8, 3 9) The energy density decreases with increase in amino acids and results in lower energy availability and exposure to UV illumination for individual amino acids during PUV treatment S H/S S interchange reactions may mask the potent ial antigenic epitopes on the surface of the molecule during reaggregation, and thus reduces the antigenicity. Thiol lactoglobulin forms larger aggregates and causes a slight change in confirmation around aromatic amino acid s and may be responsible for reduction in antigenicity (Zhong and others 2012). Peptide backbones suc h as tryptophan, tyrosine, phenylalanine and cysteine are targeted during UV induced photo degradation of proteins (Correia and others 2012) UV absorption can excite tyrosine to higher electronic energy states and leads several further processes such as r elaxation to ground state, triplet state formation, peroxy radicals formation and photochemical or photophysical processes. Tyr osine can transfer its excited state energy to tryptophan or can form the uncharged radical by ejecting an electron from the resi due (Kerwin and Remmele 2007) This tyrosine radical can involve in cross linking through the ortho positi on and forms dityrosine (Kerwin and Remmele 2007; McCormick and others 1998) Dityro s i ne is commonly found in exposure to oxygen free radicals, nitrog en dioxide, UV radiation and gamma radiation. Dityrosine molecule can be either intermolecular or intramolecular and is one of the specific markers for protein oxidation.
48 Protein aggregation in PUV treated sample may be the result of this intermolecular cr oss linking. Reduction of disulfide bridges (S S) is another key photoc hemical mechanism that follows t yr osine excitation (Neves Petersen and others 2009) Photoionization or biphotonic absorption in the triplet state generates solvated electrons that ca n be captured by cysteines and leads to the formation of RSSR and S S breakage (Hoffman and Hayon 1972) The ion d isulfide can also undergo protonation that leads to S S disruption. Excitation of cysteine residue at 254 nm can also result in photolysis of S S with formation of RSSR lactalbumin, cutinase and lysozyme lost S S bond after illuminating with UV light (Correia and others 2012) The presence of lactoglobulin and lactalbumin makes whey protein more susceptible to PUV illumination and yields higher antigen mitigation S casein which does not have any disulfide bond Presence of any photosensitizer can cause photosensitizing reactions such as the presence of riboflavin for photooxidation. Heat sensitivi ty of whey protein and possible photochemical reactions during PUV treatment suggests the photochemical and photothermal effect in mitigating antigenicity. Further studies have to be conducted to understand the photophysical effect on proteins. Indirect E LISA The quantification of IgE binding was evaluated with indirect ELISA. Plasma pooled from three sensitive individual s with a documented history of milk protein allergenicity was used for analysis. Figure 3 5 shows IgE binding capacity of PUV treated whe casein proteins. The samples were significantly different ( )
49 from each other and with control. The energy absorbed by the sample is increased with an increase in treatment time and hence, the most antigen reduction was observed at 180 s. The energy absorbed by protein may lead to conformational and structural changes, which can alter functions and IgE binding ability of proteins. A s imilar phenomenon was observed with other types of processing techno log ies such as gamma radiation. Allergenic ty was decreased with an increase in irradiation dose on milk proteins (Lee and others 2001). The author suggested that conformational changes to protein and structural changes to epitope were responsible for the reduction in allergenicity. PUV is also exp ected to have similar effect s on protein structure. There was a significant difference in the IgE binding for PUV treated whey and casein proteins. Depending upon the amino acid composition and molecular structure, proteins may exhibit different responses with UV radiation (Gennadios and others 1998). Hence, the degree of reduction in antigenicity of these proteins can be different and the similar results were observed in our IgE binding experiments T he temeprature of the sample increased by 40 o C after 6 0 s treatment at a distance of 10.2 cm. According to Lee (1992), casein is very stable to heat and it can withstand 130 o C for one hour without coagulation. This confirms the minimal thermal casein even at higher tr eatments such as 180 s. However, the synergistic thermal effect during PUV treatment cannot be undermined. Based on the results, 180 s treatment can be considered as the optimum treatment condition for PUV treatment to mitigate antigenicity of milk protein s. However, further in vivo nutritional and quality studies have to be performed to evaluate the effect of PUV treatment.
50 Other B enefits As PUV is shown to be effective in mitigating antigenicity, there is a potential to develop hypo allergic food product s using PUV treated milk as an ingredient. Krishnamurthy and others (2005) applied PUV on raw milk to inactivate by 8.55 log cfu/ml reduction S taphylococcus aureus and extend the possible application of PUV for milk pasteurization. Hence, PUV can be used a s a tool to inactivate pathogens in milk while mitigating allergens and further reduces operating costs. Employing these techniques can also reduce the treatment time as there is no preheating or cooling required as opposed to heat pasteurization. In the w avelength region of 280 to 310 nm, 7 dehydrocholesterol functions as a precursor for vitamin D3 and milk is inherently a good source of 7 dehydrocholesterol. Marry (2009) o bserved an increase in vitamin D2 after treating white button mushrooms with PUV. Fu rther research has to be conducted to confirm the conversion of 7 dehydrocholesterol to vitamin D3 in milk, when treated with PUV.
51 Figure 3 1. SDS PAGE for whey proteins showing decrease in band intensitie s of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatment for 180 s
52 Figure 3 2. SDS PAGE for casein sho wing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatment for 180 s
53 Fig ure 3 3 Wes tern blot for whey protein showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatment for 180 s
54 Fig ure 3 4 Western blot for casein showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatment for 180 s
55 Figure 3 5. Indirect ELISA results for PUV treated isolated milk proteins indicating reduction in IgE binding after PUV treatment 0 10 20 30 40 50 60 70 80 90 100 0 s 60 s 120 s 180 s IgE binding ( % B/B 0 ) sample Whey casein
56 Figure 3 lactoglobulin ( Source : www.uniprot.org/uniprot/P02754 ) 1 0 2 0 3 0 4 0 5 0 6 0 MKCLLLALAL TCGAQALIVT QTMKGLDIQK VAGTWYSLAM AASDISLLDA QSAPLRVYVE 7 0 8 0 9 0 10 0 11 0 12 0 ELKPTPEGDL EILLQKWENG ECAQKKIIAE KTKI PAVFKI DALNENKVLV LDTDYKKYLL 13 0 14 0 15 0 16 0 17 0 FCMENSAEPE QSLACQCLVR TPEVDDEALE KFDKALKALP MHIRLSFNPT QLEEQCHI
57 Figure 3 lactalbumin ( Source : www.uniprot.org/uniprot/P00711 ) 1 0 2 0 3 0 4 0 5 0 6 0 MMSFVSLLLV GILFHATQAE QLTKCEVFRE LKDLKGYGGV SLPEWVCTTF HTSGYDTQAI 7 0 8 0 9 0 10 0 11 0 12 0 VQNNDSTEYG LFQINNKIWC KDDQNPHSSN ICNISCDKFL DDDLTDDIMC VKKILDKVGI 13 0 14 0 NYWLAHKALC SEKLDQWLCE KL
58 Figure 3 s2 casein (S ource: www.uniprot.org/uniprot/P02663 ) 1 0 2 0 3 0 4 0 5 0 6 0 MKFFIFTCLL AVALAKNTME HVSSSEESII SQETYKQEKN MAINPSKENL CSTFCKEVVR 7 0 8 0 9 0 10 0 11 0 12 0 NANEEEYSIG SSSEESAEVA TEEVKITVDD KHYQKALNEI NQFYQKFPQY LQYLYQGPIV 13 0 14 0 15 0 16 0 17 0 18 0 LNPWDQVKRN AVPITPTLNR EQLSTSEENS KKTVDMEST E VFTKKTKLTE EEKNRLNFLK 19 0 20 0 21 0 22 0 KISQRYQKFA LPQYLKTVYQ HQKAMKPWIQ PKTKVIPYVR YL
59 Figure 3 s1 casein ( S ource: www.uniprot.org/uniprot/P02662 ) 1 0 2 0 3 0 4 0 5 0 6 0 MKLLILTCLV AVALARPKHP IKHQGLPQEV LNENLLRFFV APFPEVFGKE KVNELSKDIG 7 0 8 0 9 0 10 0 11 0 12 0 SESTEDQAME DIKQMEAESI SSSEEIVPNS VEQKHIQKED VPSERYLGYL EQLLRLKKYK 13 0 14 0 15 0 16 0 17 0 18 0 VPQLEIVPNS AEERLHSMKE GIHAQQKEPM IGVNQELAYF YPELFRQFYQ LDAYPSGAWY 19 0 20 0 21 0 YVPLGTQYTD APSFSDIPNP IGSENSEKTT MPLW
60 CHAPTER 4 EFFECTS OF PULSED ULTRAVIOLET L IGHT AND HIGH HYDROS TATIC PRESSURE ON THE ANTI GENI CITY OF ISOLATED EGG PROTEINS Background Food allergies are important health problems in industrialized countries, with approximately 2% of the adult population and 8% of children affected by food allergies ( Monaci and others 2006), yet symptoms are exhibi ted among 22% of the general population (Woods and others 2002). The annual incident rate (new cases/year) of sensitization to food allergens such as egg, wheat, milk and soy decreases from 10% at one year of age to 3% at six years of age (Mills and Breite neder 2005). Food of plant origin such as peanuts, tree nuts, wheat and soybean and of animal origin such as minute amounts of inflicted food can cause allergy in the se nsitive individuals. Besides milk and peanut allergies, egg allergy has become the most frequent food allergies in westernized countries. Egg allergy is the second leading cause of food allergy in children with an estimated pr evalence of 1.6% to 3.2% (Mine and Yang 2008). A dose sensitive individuals, while a dose of 0.15 mg can do 1 in 100 (Anonymous 2004). 11% shell, 56 61% white and 27 32% yolk Egg white is an aqueous protein solution of 10% protein and 88% water. Egg yolk consists of 50% water, 34% lipid and 16% protein (Poulsen and others 2001). Five proteins in egg white and egg yolk are characterized as major allergens. As the major allerge n source, egg white contains four major allergens: 11% of ovomucoid (gal d 1, 28kDa), 55% of ovalbumin (gal d 2, 43 kDa), 12% of ovotransferrin (gal d 3, 78 kDa) and 3% of lysozyme (gal d 4, 14kda) (Heine and others 2006). The immune dominant protein in
61 eg g white is ovomucoid (glycoprotein) that comprises 186 amino acids (Bernhisel Broadben and others 1994). Chicken serum albumin (gal d 5, 69 kDa) is the major allergen present in egg yolk. An allergic reaction is initiated when an Immunoglobulin E (IgE) ant ibody binds to a specific epitope located on the allergen. Epitopes can be either sequential, where a specific sequence of amino acid is recognized, or conformational, which is dependent on tertiary struct ure of the target protein (Lin and Sampson 2009). P rotein antigenicity can be altered during food processing that can destruct conformational epitopes, but processing may have limited effect on sequential epitopes. Chemical reactions between proteins, fats, and sugars in food matrix may alter the conformat ion of allergen epitopes, causing the epitopes to become masked to the immune response. Antigenicity of food proteins can be modified with Maillard reaction, enzymatic browning or roasting and other dry heating processes. Neoepitopes formed during food pro cessing can increase or decrease antigenicity (Anna and Fiocchi 2009). Most egg allergic individuals are sensitive to both raw and cooked eggs. In very few cases, individuals who are sensitive to raw egg can tolerate cooked egg. Removal of epitopes on egg allergens by enzyme fragmentation may reduce the antigenicity in egg. Heating and freeze drying do not reduce the antigenicity to a safer level in eggs (Wal, 2004). Although the complete avoidance of eggs or egg containing foods is a key to prevent egg al lergies, such a practice is not always possible, since egg is a common food ingredient that is ubiquitous. Undeclared allergens present in packaged foods can cause severe health problems to sensitized individuals.
62 When ovalbumin, lysozyme, ovotransferrin and ovomucoid were heat treated for 15 min at 95 o C a decrease in human IgE binding activity was observed for ovom ucoid and ovotransferrin (Mine and Zhang 2002). The carboxymethylation of ovomucoid and ovotransferrin reduced binding activity. Previous stu dies found that antigenicity of ovomucoid, ovotransferrin and lysozyme was decreased whereas ovalbumin was not affected by carboxymethylation. Ovalbumin, lysozyme and ovotransferrin had higher human IgE binding activity with urea treat ment compared to cont rol (Mine and Zhang 2002). Heated and ovomucoid depleted egg white was more hypoallergenic than heated or freeze dried egg white (Urisu and others 1997). Therefore, a more ideal strategy against food allergy is to destroy or minimize allergens during proce ssing before foods reach the consumer. Pulsed ultraviolet light (PUV) is an emerging techno log y that has been reported in literature to be effective in reducing peanut (Yang and others 2011a; Chung and others 2008), almond (Li and others 2011), shrimp (Shr iver and others 2011a,b; Yang and others 2012), soybean (Yang and others 2011b) and wheat allergens (Nooji and others 2011). In this technique, a capacitor stores electrical energy and releases the energy as short period pulses through a lamp filled with a n inert gas. It causes ionization of gas and produces broad spectrum of light having wave lengths from ultraviolet to near infrared in the region of 100 to 1100 nm. Ultraviolet (UV), visible and infrared regions account for 54, 26 and 20% of the PUV energy respectively. Ultraviolet light can ionize molecules due to its high energy contribution. Visible and infrared lights result in vibration and rotation of molecules, respectively (Krishnamurthy and others 2009). Keklik and others (2009) successfully utili ze PUV for decontamination of shell eggs.
63 High hydrostatic pressure (HHP) uses 100 1000 MPa pressure either to change the food attribute or inactivate microorganisms with or without supplemental heat (Balasubramaniam and Farkas, 2008). Usually, the treat ment is carried out in a pressure vessel using water as the pressure transmitting medium. Pressure is applied in all directions for a desired holding (treatment) time and then released. This isostatic pressure helps the solid sample retain its shape. Micro bial inactivation can be achieved at lower pressures compared to enzyme inactivation, and treatment conditions depend on the sample and expected final results (San Martin and others 2002). The HHP treated foods are microbio log ically stable and yet retain f ood quality and freshness as HHP does not affect covalent bo nds (Balasubramaniam and Farkas 2008). The HHP techno log y has been used in industry for improving the shelf life and microbial safety of food products, and its application for solute diffusion pro cess, freeze thawing process and functional property modification of proteins have also been tested (San Martin and others 2002). It has been reported that HHP reduces the antigenicity of lactoglobulin and ovalbumin (Iametti and others 1999; Kleber and others 2007), but does not affect that of the almond protein extracts (Li and others 2011). The major objective of this study was to examine the efficacy of PUV and HHP treatments in reduci ng the antigenicity of isolated egg proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), western blot, and indirect enzyme linked immune sorbent assay (ELISA) techniques. Materials and Methods The assay plasma, pooled from f our patients with documented history of egg allergy was purchased from Plasma Lab International, Everette, WA. Mouse anti chain specific) conjugated to horseradish peroxidase (HRP) was
64 purchased from Invitrogen, Camarillo, CA. Grade A large eggs were purchased from a local store (Publix, Gain esville, FL). Avure lab scale high pressure processor (model PT 1, Kent, WA) monitored with DASYLab 7.0 Software (DASYTEC USA, Bedford, NH) was used for the HHP treatments and Xenon Steripulse XL 3000 batch PUV system (Xenon Corporation, Woburn, MA) was used for the PUV treatments. W hole eggs were homogenized by stirring on a magnetic stirrer for 10 min at room temperature. The homogenate was suspended in sodium phosphate buffered saline solution (PBS at pH 7.4) at 1:1 volume ratio and stirred overnight a t room temperature. The insoluble residues were removed by centrifugation at 2500 g for 10 min. Five ml protein solution was placed in an aluminum dish and subjected to PUV treatments at a constant distance of 10.2 cm from the quartz window of the batch X enon PUV sterilizer. The samples were exposed to PUV for 60, 120 and 180 s. For the HHP treatment, 1.5 ml of isolated egg protein solution was transferred to a sterile polypropylene pouch, heat sealed and enclosed in a secondary pouch to treat in an Avure lab scale high pressure processor using water as the hydrostatic medium (Model Avure PT 1, Avure Techno log ies, Kent, WA) Samples were treated at 600 MPa for 5, 15, and 30 min at three initial temperatures: 4, 21, and 70 o C Ten l of the treated sample wa s added to 300 l of Coo massie blue reagent and allowed to stand for 10 min at room temperature before measuring the absorbance at 595 nm (Spectra Max 340 PC). Proteins concentration was analyzed by fitting means of absorbance readings in the standard curv e. SDS PAGE and W estern B lot Pre cast gels (Bio Rad, Hercules, CA) were used to run electrophoresis with PowerPacTM HC. Protein structure was unfolded by heating 10 l of the sample + 10 l
65 of XT buffer 2X on hot water bath at 100 o C for 10 min. Ten g of protein was loaded in each well of 4 20% of Tris HCl gel and run for 1 h at 160 V. GelcodeTM Blue safe protein stain solution (Pierce) was used for staining the gels for 45 min and then the gels were de stained for 90 min using deionized water. Gel was run in a similar procedure as described above for western blot. The gel was placed in a transfer buffer for 10 min after electrophoresis. It was then transferred to an Immobilon P (Millipore) membrane by running at 10 V for 30 min using Trans Blot SD cell. M embrane was blocked with blocking buffer for 2 h at room temperature. TBS containing 0.05% Tween 20 (TBST) was used to wash transfer membrane after each step. A 1:5 ratio of primary antibody solution was used to incubate transfer membrane overnight, follow ed by incubating for 2 h in a 1:250 ratio of secondary antibody solution. The transfer membrane was then added with one step chloronaphthol to observe IgE binding. Indirect ELISA IgE binding of treated samples was also examined by the Indirect ELISA. One h ml concentrated samples were applied on each well and incubated for 2 h at 37 o C. Tris buffer saline containing 0.05% Tween 20 (TBST) was used to wash plates for three times after incubation, and the plate was patted each time o n prepared mat. One hundred microliters of superblock was applied to each well and incubated for 2 h at room temperature for blocking. The plates were washed again three as sera were added to untreated sample for the comparison. The plates were washed for three times anti human IgE IgG solution was added to each well and incubated for 1 h at room
66 tempe rature. The plates were then washed for three times. One tab of 5 mg o phenylenediamine dihydro chloride (OPD) was dissolved in 9 ml of water and 1 ml of stable peroxide buffer to prepare substrate and 100 l of this substrate was added to each well for co lor reaction. One hundred micro liter of 2.5 M H 2 SO 4 was added after 20 min to stop the reaction and color change from clear to golden yellow was observed. The spectra Max 340 PC was used to measure the absorbance at 490 nm Statistical Analysis Statistica l analysis was performed using SAS software (SAS Institute Inc., Cary, NC, USA) using analysis of variance (ANOVA). was used for comparison of means. Results and Discussion SDS P AGE and W estern B lot for PUV T reated S amples E lec trophoresis for the isolated egg protein is reported in Fig ure 4 1. Lysozyme, ovalbumin, chicken serum albumin and ovotransferrin proteins are the major allergens present in egg and the bands associated with each protein appeared at 14, 43, 69 and 78 kDa, respectively (second lane). Protein electrophoretic profiles from lane 3 showed that band int ensities of these allergens decreased with 60 s of PUV treatment. The IgE binding of these egg proteins was observed using Western blot with pooled human pl asma of egg allergi c patients. The IgE recognized lysozyme (14 kDa), ovalbumin (43 kDa) and chicken serum albumin (69 kDa) in the control sample. However, IgE binding was greatly reduced in PUV treated egg proteins. Lane 5 (Figure 4 2), which represents isolated egg proteins treated for 180 s, showed significant decrease in IgE binding of these allergens.
67 Western blot (Figure 4 2) showed higher IgE binding reduction of lysozyme compared to ovalbumin. According to Mine and Zhang (2002), ovomucoid and lysozyme may contain both sequential and conformational IgE epitopes, whereas ovalbumin contains sequential IgE epitopes. Ovotransferrin, lysozyme and chicken serum albumin have more UV susceptible disulfide bonds compared to ovalbumin (Figure 4 7, 4 8, 4 9, 4 10). Depending upon the amino acid composition and molecular structure, proteins may exhibit different responses to UV radiation (Gennadios and others 1998). Hence, PUV was more effective against lysozyme than ovalbumin. Sequential epitopes are more stab le during food process ing (such as mechanical, irradiation or heat treatment), while conformational epitopes can be denatured (Yada 2004). Pulsed UV light might have an intense effect on conformational epitopes compared to sequential epitopes, which result s in higher reduction of IgE binding with lysozyme. Lee and others (2005) observed the reduction in antigenicity of ovalbumin in irradiated egg white cake. Lee and others casein allergen with 3, 5 and 10 kGy doses of gamma irradiation. When a low d ose of gamma irradiati on was applied to soy proteins, intensity of the allergen band was found to decrease because of a slight breakdown in the polypeptide chain (Lee and others 2005). In another study, aggregates formed during PUV treatment of peanut prot eins decreased their solubility (Chung and others 2008). Since 54% of the total PUV energy comes from the UV fraction, which can ionize molecules, PUV is expected to exhibit a similar mechanism as gamma irradiation in reducing the antigenicity of food prot eins. Fragmentation and/or aggregation of egg proteins during PUV treatment might have resulted in lowering IgE binding. Shriver and others (2011a) stated that fragment
68 proteins might have travelled through the acrylamide gel faster and lost into the SDS l ower buffer chamber whereas aggregated proteins might have difficulties in penetrating through the gel, causing them to have a smeared appearance in SDS PAGE in the higher molecular weight region and in the Western blots. Indirect ELISA for PUV T reated S a mples Human plasma pooled from four egg sensitive individuals was used for quantifying IgE binding using indirect ELISA. The IgE binding of PUV treated proteins, control with allergenic sera and control with non allergenic sera (normal) is reported in Fig u re 4 3 The treatments of PUV treated sample compared to control indicates the presence of less antigenicity in treated sample s In the study, IgE binding was found to decrease with i ncreased treatment time during PUV processing. About 47.4, 94.8 and 144.2 J/cm 2 of energy can be absorbed by the sample when placed at 10.2 cm distance from the quartz window of this PUV lamp and treated for 60 s, 120 s and 180 s, respectively (Krishnamurt hy, 2006). Optical properties and presence of solids in the sample play s a vital role in energy absorption during processing. Treated sample was transparent and thickness of the sample was maintained at < 5 mm to maximize energy absorption. This high amoun t of energy can cause conformational and structural changes, thus reducing the IgE binding. Proteolysis that can destroy conformational epitopes, can unmask sequential (linear) epitopes. Sequential epitopes that were masked with protein hydrophobic doma in and/or three dimensional native structure can be exposed with proteolysis (Haddad and others 1979; Wal 2004). Hence, protein hydrolysates are used in hypoallergenic produc ts since hydrolysis reduces allergenicity. The p
69 structure and str ucture of attached side chains such as carbohydrates lead to antigenicity, hence changing these structures can lower the antigenicity (Lee 1992). Super radicals formed by UV light can induce carbohydrate linkage, protein crosslinking, and pr otein fragmenta tion (Kolakowska 2003). PUV is expected to induce similar effects on proteins, and these effects might have resulted in reduction of the antigenicity of PUV treated isolated egg protein. Cost C omparisons of PUV T reatment Pulsed UV light is an emerging tec hno log y which is also effective in inactivating surface pathogens at lower treatement times. A 5.3 log reduction (cfu /cm 2 ) of Salmonella Enteritidis was observed in egg with 23.6 + 0.1 J/cm 2 energy of PUV ( Keklik and others 2009) Based on the results for an tigenicity mitigation in this study and the literature on PUV inactivation of pathogens, it can be deduced that PUV may be utilized for pathogens and allergen inactivation simultaneously, while preserving the quality of egg. Employing PUV can also potentia lly reduce the operational costs as both pathogens and allergens in eggs can be inactivated altogether by these techniques. Although no actual economic study was conducted here a comparison is made based on the published data from the literature It cost s 0.4 1.25, 25 /m 3 for reducing E. coli by 4 log cfu/ml 10 in primary waste water using UV light, electron beam amd gamma irrad iation, respectively (Taghipour 2004). PureBright PUV system will cost 1.1 /m 2 (0.1 /ft 2 ) for 4 J/cm 2 energy treatment (Dunn and othe rs 1997). Electricity, maintenance and equipment amortization are included in this cost estimation (Dunn and others 1997). Capital and operational costs can be reduced as PUV can inactivate pathogens and allergens in egg at the same time under the same tre atment conditions.
70 Effect of PUV on S ample T emperature and M oisture The PUV treatment for 180 s resulted in 50% moisture loss in the sample. According to Chung and others (2008), 40% of moisture loss was observed during PUV treatment of 10 ml peanut protei n sample. With increased treatment time, energy absorbed by the sample increased and the highest moisture loss was observed for 180 s treatment s Temperature of the sample was increased by 40 o C for the same treatment. Since infrared contributes to 20% of PUV radiation, this energy was responsible for the temperature rise during the PUV treatment. After molecules are excited upon absorbing energy, they can come back to ground state either by releasing energy as heat or photons or by inducing some chemical c hanges (Krishnamurthy and others 2009). When a sample is exposed to light, a portion of the light is transmitted and the rest is reflected. The intensity of transmitted light while penetrating through the sample exponentially decays and gets converted to t hermal energy. Conduction transfers this heat to inner layers of the sample (Palmieri and others 1999). Depending on temperature, time, intrinsic characteristics of the protein and physicochemical conditions of its environment, thermal treatment caus es significant changes in protein structure (Anonymous 2004). Proteins have physical transformations such as loss of secondary structure, cleavage of disulfide bonds, formation of new intra /inter molecular interactions, and formation of aggregates at 55 7 0 o C 70 80 o C 80 90 o C and 90 100 o C temperatures, r espectively (Davis and Williams 1998). The temperature of the sample attained was 62 o C at 180 s treatment. This temperature increased with increased treatment time. Heat denaturation of protein can res ult in tertiary structure alteration. Further digestion in gastrointestinal tract causes alteration
71 and break up of the tert iary structure (Lin and Sampson 2009). Altered tertiary structure changes conformational epitopes that are recognized by IgE antibod ies. Thus, the rise in temperature (heat energy) during PUV treatment also affects antigenicity of proteins. make up complicate the thermal processing in changing the antig enicity of protein s (Davis and others 2001). Effect of H igh H ydrostatic P ressure After treating the samples with HHP at 600 MPa for 5, 15 and 30 min at three initial temperatures of 4, 21 and 70 o C they were analyzed for protein electrophoretic profiles a nd IgE binding SDS PAGE (Figure 4 4) did not show any change in band intensities of maj or allergens. Western blot (Figure 4 5) showed a decrease in IgE binding of these allergens when treated at 21 o C However, indirect ELISA (Figure 4 6) showed an increa se in overall IgE binding of the sample s When cod and mackerel muscle was subjected to HHP, certain extractable protein bands disappeared in SDS PAGE (Ohshima and others 1993). Ohshima and others (1993) suggested that sarcoplasmic proteins had covalent li nkage rather than degradation, which resulted in resistance to extraction with SDS. Ultrahigh pressure might have resulted in protein aggregates that still had antigenicity as recognized in indirect ELISA but not in Western blot, since the large sized prot ein aggregates were unable to migrate through the gel. Hence, there were no bands in SDSPAGE and Western blot though antigenicity of the protein was increased as shown in indirec t ELISA. These results suggest the ineffectiveness of HHP in mitigating egg a llergens under the tested conditions. Li and others (2011) showed that HHP did not have any effect on the antigenicity of almond protein extracts when they were treated at the same conditions as tested in this study.
72 However, L ametti and others (1999) red uced antigenicity of ovalbumin by 40% lactoglobulin decreased when treated at 200, 400 and 600 MPa for 10 and 30 min at 60 o C and 68 o C (Kleber and others 2006). When high pressure was applied on lactoglobulin at room temperature, secondary and tertiary protein structures were altered significantly (Walker and others 2004). It is speculated that reducing structural integrity and increasing accessibility to digestive enzymes due to unfolding may lo wer the antigenicity of high lactoglobulin (Zeece and others 2008). However, lower temperatures such as 40 o C and 50 o C lactoglobulin (Kleber and others 2006). Polyp henoloxidase enzyme activity increased in mu shrooms after 400 MPa treatment, in potatoes after 200 600 MPa treatment and in apples after 200 600 MPa treatment. However, 800 MPa treatment resulted in complete inactivation (Gomes and Ledward 1996). Hence, the effect of HHP on enzymes/proteins depe nded on treatment time, pressure applied and type of enzyme/protein. More studies (with different time and pressure conditions) need to be conducted to further examine the efficacy of HHP on allergen mitigation and for its application to reduce the antigen icity of isolated egg proteins.
73 Fig ure 4 1. SDS PAGE for egg proteins showing decrease in band intensities of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatmen t for 180 s 10 20 15 25 50 37 75 250 150 100 kDa Ovalbumin 43 kDa Ovotransferrin 78 kDa Lysozyme 14 kDa Chicken Serum Albumin 69 kDa
74 Figure 4 2. Western blot for egg proteins showing decrease in IgE binding of major allergens after PUV treatment. (M) Marker, (C) Control, (T1) PUV treatment for 60 s, (T2) PUV treatment for 120 s, and (T3) PUV treatment for 180 s 10 20 15 25 50 37 75 250 150 100 kDa Lysozyme 14 kDa Ovalbumin 43 kDa Chicken Serum Albumin 69 kDa
75 Figure 4 3. Indirect ELISA results for PUV treated isolated egg proteins indicating reduction in IgE binding after PUV treatment. Treatments not connected by same letter are significantly different from control A B C
76 Figure 4 4 SDS PAGE for egg pr oteins showing band intensities of major allergens after HHP treatment. (M) Marker, (C) Control, (H1) at 4 o C for 5 min, (H2) at 4 o C for 15 min, (H3) at 4 o C for 30 min, (H4) at 21 o C for 5 min, (H5) at 21 o C for 15 min, (H6) at 21 o C for 30 min, (H7) at 70 o C for 5 min and (H8) at 70 o C for 15 min
77 Figure 4 5. Western blot for egg proteins showing decrease in IgE binding of major allergens after HHP treatment. (M) Marker, (C) Control, (H1) at 4 o C for 5 min, (H2) at 4 o C for 15 min, (H3) at 4 o C fo r 30 min, (H4) at 21 o C for 5 min, (H5) at 21 o C for 15 min, (H6) at 21 o C for 30 min, (H7) at 70 o C for 5 min and (H8) at 70 o C for 15 min C H1 H2 H3 H4
78 Figure 4 6 Indirect ELISA results for HHP treated isolated egg proteins indicating increase in IgE bindin g after HHP treatment Treatments not connected by same letter are significantly different from control A C B
79 Figure 4 7. Amino Acid sequence for ovalbumin (S ource: www.uniprot.org/uniprot/P01012 ) 1 0 2 0 3 0 4 0 5 0 6 0 MGSIGAASME FCFDVFKELK VHHANENIFY CPIAIMSALA MVYLGAKDST RTQINKVVRF 7 0 8 0 9 0 10 0 11 0 12 0 DKLPGFGDSI EAQCGTSVNV HSSLRDILNQ ITKPNDVYSF SLASRLYAEE RYPILPEYLQ 13 0 14 0 15 0 16 0 17 0 18 0 CVKELYRGGL EPINFQTAAD QARELINSWV ESQTNGIIRN VLQPSSVDSQ TAMVLVNAIV 19 0 20 0 21 0 22 0 23 0 24 0 FKGLWEKAFK DEDTQAMPFR VTEQESKPVQ MMYQIGLFRV ASMASEKMKI LELPFASGTM 25 0 26 0 27 0 28 0 29 0 30 0 SMLVLLPDEV SGLEQLESII NFEKLTEWTS SNV MEERKIK VYLPRMKMEE KYNLTSVLMA 31 0 32 0 33 0 34 0 35 0 36 0 MGITDVFSSS ANLSGISSAE SLKISQAVHA AHAEINEAGR EVVGSAEAGV DAASVSEEFR 37 0 38 0 ADHPFLFCIK HIATNAVLFF GRCVSP
80 Figure 4 8. Amino Acid sequence for chicken serum albumin (S ource: www.uniprot.org/uniprot/P19121 ) 1 0 2 0 3 0 4 0 5 0 6 0 MKWVTLISFI FLFSSATSRN LQRFARDAEH KSEIAHRYND LKEETFKAVA MITFAQYLQR 7 0 8 0 9 0 10 0 11 0 12 0 CSYEGLSKLV KDVVDLAQKC VANEDAPECS KPLPSIILDE ICQVEKLRDS YGAMADCCSK 13 0 14 0 15 0 16 0 17 0 18 0 ADPERNECFL SFKVSQPDFV QPYQRPASDV ICQEYQDNRV SFLGHFIYSV ARRHPFLYAP 19 0 20 0 21 0 22 0 23 0 24 0 AILSFAVDFE HALQSCCKES DVGACLDTKE IVMREKAKGV SVKQQYFCGI LKQFGDRVFQ 25 0 26 0 27 0 28 0 29 0 30 0 ARQLIYLSQK YPKAPFSEVS KFVHDSI GVH KECCEGDMVE CMDDMARMMS NLCSQQDVFS 31 0 32 0 33 0 34 0 35 0 36 0 GKIKDCCEKP IVERSQCIME AEFDEKPADL PSLVEKYIED KEVCKSFEAG HDAFMAEFVY 37 0 38 0 39 0 40 0 41 0 42 0 EYSRRHPEFS IQLIMRIAKG YESLLEKCCK TDNPAECYAN AQEQLNQHIK ETQDVVKTNC 43 0 44 0 45 0 46 0 47 0 48 0 DLLHDHGEAD FLKSILIRYT KKMPQVPTDL LLETGKKMTT IGTKCCQLGE DRRMACSEGY 49 0 50 0 51 0 52 0 53 0 54 0 LSIVIHDTCR KQETTPINDN VSQCCSQLYA NRRPCFTAMG VDTKYVPPPF NPDMFSFDEK 55 0 56 0 57 0 58 0 59 0 60 0 LCSAPAEERE VGQMKLLINL IKRKPQMTEE QIKTIADGFT AMVDKCCKQS DINTCFGEEG 61 0 ANLIVQSRAT LGIGA
81 Figure 4 9. Amino Acid sequence for ovotransferrin (S ource: www.uniprot.org/uniprot/P02789 ) 1 0 2 0 3 0 4 0 5 0 6 0 MKLILCTVLS LGIAAVCFAA PPKSVIRWCT ISSPEEKKCN NLRDLTQQER ISLTCVQKAT 7 0 8 0 9 0 10 0 11 0 12 0 YLDCIKAIAN NEADAISLDG GQAFEAGLAP YKLKPIAAEV YEHTEGSTTS YYAVAVVKKG 13 0 14 0 15 0 16 0 17 0 18 0 TEFTVNDLQG KTSCHTGLGR SAGWNIPIGT LLHRGAIEWE GIESGSVEQA VAKFFSASCV 19 0 20 0 21 0 22 0 23 0 24 0 PGATIEQKLC RQCKGDPKTK CARNAPYSGY SGAFHCLKDG KGDVAFVKHT TVNENAPDQK 25 0 26 0 27 0 28 0 29 0 30 0 DEYELLCLDG SRQPVDNYKT CNWARVAAHA VVARDDNKVE DIWSFLSKAQ SDFGVDTKSD 31 0 32 0 33 0 34 0 35 0 36 0 FHLFGPPGKK DPVLKDLLFK DSAIMLKRVP SLMDSQLYLG FEYYSAIQSM RK DQLTPSPR 37 0 38 0 39 0 40 0 41 0 42 0 ENRIQWCAVG KDEKSKCDRW SVVSNGDVEC TVVDETKDCI IKIMKGEADA VALDGGLVYT 43 0 44 0 45 0 46 0 47 0 48 0 AGVCGLVPVM AERYDDESQC SKTDERPASY FAVAVARKDS NVNWNNLKGK KSCHTAVGRT 49 0 50 0 51 0 52 0 53 0 54 0 AGWVIPMGLI HNRTGTCNFD EYFSEGCAPG SPPNSRLCQL CQGSGGIPPE KCVASSHEKY 55 0 56 0 57 0 58 0 59 0 60 0 FGYTGALRCL VE KGDVAFIQ HSTVEENTGG KNKADWAKNL QMDDFELLCT DGRRANVMDY 61 0 62 0 63 0 64 0 65 0 66 0 RECNLAEVPT HAVVVRPEKA NKIRDLLERQ EKRFGVNGSE KSKFMMFESQ NKDLLFKDLT 67 0 68 0 69 0 70 0 KCLFKVREGT TYKEFLGDKF YTVISSLKTC NPSDILQMCS FLEGK
82 Figure 4 10. A mino Acid sequence for lysozyme (S ource: www.uniprot.org/uniprot/P00698 ) 1 0 2 0 3 0 4 0 5 0 6 0 MRSLLILVLC FLPLAALGKV FGRCELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA 7 0 8 0 9 0 10 0 11 0 12 0 TNRNTDGSTD YGILQINSRW WCNDGRTPG S RNLCNIPCSA LLSSDITASV NCAKKIVSDG 13 0 14 0 NGMNAWVAWR NRCKGTDVQA WIRGCRL
83 CHAPTER 5 ANTIOXIDANT CAPACITI ES AND HEALTH ENHANC ING PHYTONUTRIENT CONTENTS OF SOUTHERN HIGH BUGH BLUEBERRY WINE COMPARED TO GRAPE WINES AND FRUI T LIQUORS Background Blueberry ( Vaccini um spp .) is a popular natural food product consumed worldwide. For centuries, Native American tribes have used the leaves, roots, and fruits from the blueberry plant for medicinal purposes ( Sanchez Moreno and others 2003) and blueberries continue to be us ed in many types of dietary health products as pharmaceutical or food supplements ( Kal t and Dufour 1997 ). In the U.S., blueberries rank only second to strawberries in terms of berry consumption, while in Canada, blueberry is the largest fruit crop grown na tionally, accounting for over half of entire Canadian fruit acreage ( Scrivener 2008 ). In North America, the most commonly cultivated species is the (Northern) highbush blueberry, but with adaption to the Southern U.S. climate, its hybrid the Southern highb ush blueberry, which was developed by crossing northern highbush varieties from Michigan and New Jersey with wild blueberries native in Florida and other southeaster n states (Williamson and Lyrene 2004), has been created and quickly spread to Florida, Geor gia, California, and even the Mediterranean regions of Europe, Southern Hemisphere countries and China. According to the U.S. blueberry (cultivated and wild) production and utilization data (USDA, 2009), of the total utilized production of 204 million tons in 2009, 101 million tons were for fresh use, while 103 million tons were processed, including blueberry wine production. Since blueberries are on the top of most everyday fruits grown or marketed in North America in terms of antioxidant capacities (USDA, 2010), blueberry wines also
84 contain significant amounts of phenols and has significantly higher antioxidant activity compared to many other wine products ( Saucier and others 1999). Antioxidants are critical to health protection by their ability to scaveng e the free radicals that can cause damage to cellular structures or DNA. Literature shows a multitude of health benefits of blueberries and blueberry wine. Due to the presence of anthocyanin, blueberries can considerably improve cardiovascular health. The anthocyanin and resveratrol contents in blueberry, which have natural cardiovascular protective qualities, are comparable or higher than red wine (Sanchez morendo and others 2003). Also, blueberry wine has marginally higher antioxidant ability than red win e and much higher antioxidant capacity than white wine (Sanchez morendo and others 2003), so b lueberry wine may be more beneficial than red wine in preventing heart diseases. Literature shows that blueberries have improved the learning capacity and motor s kills in aging animals, thus reducing the chances of dementia or and others 1999; Shukitt Hale and others 1999; Greenwell 2000; Robert and others 1977). Research shows blueberries can improve day or nighttime vision, reduce the restorat ion time after exposure to glare, and prevent weakness of eyes (macular degeneration) arising from aging. The various phytonutrients present in blueberries, such as anthocyanin, flavonoids and phenolics, part of which are carried forward to wine during fer mentation, are known for their benefits to the eyes and can help protect 2000; Robert and others 1977).
85 proanthocyanidins, ellagitannins, etc.), stilbenoids, lignans and triterpenoids, present in blueberries are capable of inhibiting cell proliferation in human breast, colon and ovarian cancer s (Damianaki and others 2000; Seeram and others 2008). Highly beneficial to the digestive and excretory system, antioxidants in blueberries help combat free radicals that can cause inflammation on the digestive pathways, and thus prevents occurrence of pe ptic ulcers ( Watson 2001 ) Eating blueberries, drinking blueberry juice, or taking blueberry wine in an appropriate quantity may help heal the existing ulcer s or hemorrhoid condition s (Seeram and others 2008). Blueberries have been found to offer protecti on from urinary tract infections caused by bacteria adhering to the mucosal lining of the bladder or urinary tract ( Ofek and others 1991; Jepson and Craig 2007 ). Blueberry wines are usually produced by fermentation through nontraditional methods. During bl ueberry wine processing, an initial press of the berries provides the juice used for fermentation along with its skin and seeds. Sulfur dioxide and pectin enzymes are usually added. After the primary fermentation, usually 2 3 weeks, wine is separated from the dregs on a free run basis and the residuals are lightly pressed to extract the remaining wine. During fermentation, phenolic compounds including catechins and other components, flavonoids and anthocyanins are transferred from the juice, skins or seeds into the blueberry wine. Su and Chien (2007) reported that the blueberry wine making process did not really lower the anthocyanin content. There are only a few studies that have quantified the antioxidant capacity, anthocyanins and total phenols in bluebe rry wines, e.g., Sanchez Moreno and others (2003), Su and Chien (2007) and Johnson and
86 others (2011). However, no analytical quantification of the foregoing antioxidant activity and phytonutrients was found in literature for the Southern highbush blueberry wine. Therefore, the objective of this study was to evaluate the antioxidant capacity and key phytonutrients of Southern highbush blueberry wine and systematically compare to grape wines and fruit liquors. Materials and Methods Southern H igh b ush B lueberry W ine S amples Southern highbush blueberry wine was obtained from a local blueberry winery for two batches of experiments conducted at the University of Florida. The blueberries used to produce the wine were a mixture of several varieties including Star, Wi ndsor, Emerald, Primadonna and Jewel. In the first batch, three 750 ml bottles were obtained for each of the different processing conditions: low sulfite, unfiltered; sulfite, unfiltered; and sulfite, filtered. Low sulfite consisted of 30 ppm total sulfite s, while normal levels of sulfite were 150 ppm total sulfites. Filtering was performed first with a crude filter (>1 ml portions of wine from each bottle were sampled for anal ysis in one la boratory, making six total observations for each wine. In a second study, 12 L of non sulfite, filtered wine, which Determination of A ntioxidant C apacity Antioxidant capacity of the Southern highbush blueberry wine was measured by the Oxygen Radical Absorbance Capacity (ORAC) method. The ORAC values were expressed as mmol Trolox equivalent (TE) per liter. For the filtered blueberry wine with no sulfite added, the ORAC values w ere determined using a modified method per Huang and others (2002). Briefly, 50 L ORAC
87 Phosphate Buffer (75 mM, ORAC PB) and appropriately diluted samples were added to a 96 well black plate (Fisher Scientific). This was followed by addition of 100 L, 20 nM fluorescein working solution to all filled wells. The mixture was incubated at 37 o C for 10 amidinopropane) dihydrochloride (140 mM, AAPH). The rate of fluorescence decay was monitored over time by calculating the area under the fluorescent decay curve and quantified using a standard curve of TROLOX, using a Spectra Max Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA). The fluorescence was monitored at 485 nm excitation an d 530 nm emissions for 40 min at 1 min intervals. The antioxidant capacities of the extracts were expressed as mmol TE/L of blueberry wine. For the other wines, the ORAC procedure was basically the same as above, except for different dilutions as: 1:1000, 1:1500, 1:2000, and 1:3000. Determination o f T otal P henols Total phenolics were determined using a modification of Fo lin To a 96 well clear plate (Fisher Scientific, Pittsburg, PA), 12.5 l of 2 N Folin phenol reagent was a dded to 50 l of deionized distilled water (ddH 2 O) and 12.5 l of wine sample. After 5 min, 7% sodium carbonate (Na 2 CO 3 ) solution (125 l) was added to the mixture and incubated (90 min, 25 o C). The absorbance of the sample was measured at 750 nm versus a reagent blank using a microplate reader. A standard curve for total phenolics was developed using gallic acid. The concentration was expressed as mg of gallic acid equivalents (GAE)/L of wine. Determination of T otal F lavonoids A standard colorimetric assay (Kim and others 2003) with slight modifications was used to quantify to tal flavonoid content. To a 96 well clear plate, 25 l of the wine
88 sample was added to 125 l of ddH 2 O. Subsequently, 7.5 l of 5% sodium nitrate (NaNO 2) was added to the mixture and allowed to stand for 5 min. Fifteen microliters of 10% aluminum chloride (AlCl 3 ) was added to the mixture and incubated at ambient temperature for an additional 5 min. Following that, 50 l of sodium hydroxide (1 M, NaOH) were added to the mixture and imme diately diluted by the addition of 27.5 l of ddH 2 O. The absorbance of the mixture was measured at 510 nm against a reagent blank and compared to a catechin standard using a microplate reader. The total flavonoids were expressed as mg of catechin equivalen ts (CE)/L of wine. Determination of T otal A nthocyanins Total anthocyanins were determined by the pH differential method (Benvenuti and others 2004). Two buffer systems, potassium chloride (KCl) (pH 1.0, 0.025 M) and sodium acetate (NaC 2 H 3 O 2 ) (pH 4.5, 0.4 M ) were utilized. An aliquot of the blueberry wine was diluted (1:10) and adjusted to pH 1.0 and pH 4.5 using the prepared buffers. Subsequently, the solutions were incubated at ambient temperature for 20 min. Absorbance was measured using a UV/VIS spectrop hotometer (Beckman Coulter, Du 730, Life Sciences UV/VIS, Lawrence, KS) at 510 nm and 700 nm at each pH, respectively. Results were calculated using E quations 5 1 5 2 and expressed as mg of cyanidin 3 glucoside/L of wine. A = (A 510nm A 700nm ) pH1.0 (A 51 0nm A 700nm ) pH4.5 ( 5 1) Anthocyanins = A ( 5 2) Where A is absorban ce, MW molecular weight [449.2] absorptivity [26,900] (Sellappan and others 2002).
89 Statistical Analysis and Comparison For the blueberry wine data obtained in this stu dy, significant differences were analyzed using 1 test by SAS version 9.0 at a 0.05 significance level. For the comparison among blueberry wines, grape wines and fruit liquors, a literature search was conducted to gather the r eported data on ORAC, total phenols and anthocyanins and tabulate them together with the measured values from this study that were referenced as a benchmark value. A percentage was calculated for the wines or fruit liquors that were above or below the benc hmark values of the blueberry wine. Results and Discussion Antioxidant C apacities, T otal P henols, A nthocyanins, F lavonoids of S outhern H igh b ush B lueberry W ine Similar to the USDA (2010) database for the ORAC values of selected foods, antioxidant capacity of the Southern highbush blueberry wine, with or without sulfite and filtration, was also determined by the ORAC method and expressed in the TROLOX equivalent (TE) values. The key phytonutrients (i.e., total phenols, total anthocyanins and total flavonoids ) were measured for the filtered blueberry wine with no sulfite added to provide the benchmark information on the level of phytonutrients of the Southern highbush blueberry wine that is eligible for labeling as organic. The results are shown in Table 5 1. The ORAC values of the Southern highbush blueberry wines ranged from 18.54 to 25.48 mmol TE/L. The ORAC values of low sulfite, unfiltered wine appeared to be significantly lower t 0.05). The average of the ORAC values shown in Table 5 1 was 22.572.92 mmol TE/L, which reflected the overall
90 antioxidant capacity of the Southern highbush blueberry wine tested in this study. This ORAC value ranked high c ompared to most blueberry wines listed in Table 5 2. According to the literature, addition of sulfite to non organic wine has two major purposes: to inhibit microorganisms and to prevent non enzymatic browning (mainly, Maillard reaction) ( Fleet 1993) Non enzymatic browning is a problem in wines that leads to a reduction in phenols, such as catechins, which are the major polyphenolic antioxidants in wines ( Saucier and Waterhouse 1999) The rate of Maillard reaction is enhanced by increasing the amount of re ducing sugars and/or increasing the temperature ( Eriksson and others 1981) Thus, if blueberry wine, which is rich in reducing sugars (10 12%) and in polyphenols, is exposed to an environment of raised temperature for an extended duration, the Maillard rea ction is favorable. As mentioned earlier, sulfite addition can combat this reaction. However, in the event of improper amount of sulfite, or in the case of aforementioned low sulfite/unfiltered wine, antioxidant capacity might diminish. Although Table 5 1 shows the effect of higher sulfite concentration on stabilizing the antioxidant activity of the blueberry wine, sulfite cannot be added to any blueberry naturally oc curring sulfite in wine. The non sulfite, filtered blueberry wine tested in this study, which could be claimed as organic, had an ORAC value of 23.49 mmol TE/L. Table 5 1 also shows the total phenolic, anthocyanin and flavonoid contents of the filtered, no n sulfite Southern highbush blueberry wine. Anthocyanins are the major water soluble flavonoids in blueberries, giving the red, purple and blue color to many fruits and vegetables (Espn and others 2007), and considered bio log ically active
91 compounds exhibi ting a wide range of health benefits, e.g., antioxidant (Cao and others 1997) antifungal (Benkeblia 2004) and anti carcinogenic properties (Ames 1983) Flavonoids are naturally occurring phenols that are present in many plants including blueberries. Flav onoids and phenolics in general are strong protector against heart disease and cancer (Yao and others 2004). The total phenols and anthocyanins of the Southern highbush blueberry wine tested in this study are compared with those of grape wines and fruit li quors in section 3.3. ORAC C omparison between Blueberry Wines a nd Grape Wines The data presented in Table 5 2 reflect the ORAC values of most blueberry and grape wines reported hitherto in the literature. A small number of wines had their antioxidant activ ities measured by a different method from ORAC, e.g., TRAP in Campos and others (1996), which were not included in Table 5 2. Grape wines are divided into red, rose and white wine types. The blueberry wines listed came from highbush or Southern Highbush va rieties. Besides the specific cultivar of Elliot and Weymouth, most blueberry wines were produced from mixed cultivars. For example, Johnson and others (2011) used 15 cultivars including Berkley, Blue Chip and Blue Haven, while in this study, the mixed cul tivars were, as mentioned earlier, Star, Windsor, Emerald, Primadonna and Jewel. To facilitate the comparison, the average ORAC value of the Southern highbush blueberry wine tested in this study, i.e., 22.57 mmol TE/L, was chosen as a benchmark. For the 20 red wines listed in Table 5 2, the ORAC values ranged from 5.25 to 39.9 mmol TE/L. Four of the 20 red wines (i.e., 20%) had ORAC values higher than 22.57 mmol TE/L, while 16 of the 20 red wines (i.e., 80%) had lower ORAC values than the Southern highbush blueberr y wine. The ORAC values of the six Rose wines listed in
92 Table 5 2 ranged from 1.52 to 11.2 mmol TE/L, which were all much lower than that the Southern highbush blueberry wine tested in this study. For the 13 white wine types listed in Table 5 2, th e ORAC values ranged from 0.6 to 5.35 mmol TE/L, which were far below that the Southern highbush blueberry wine. The much lower antioxidant potentials of white wines than red wines or blueberry wines were attributed to the fact that there is no skin or see d contact during the fermentation of white wine, while most antioxidants and phytonutrients are contained in the skins or seeds (Rigo and others 2000; Yilmaz and Toledo, 2004). The foregoing comparison suggests that blueberry wine could be more potent than red wine in health enhancement and disease prevention as far as the antioxidant capacity is concerned. Comparison of Total Phenols and Anthocyanins a mong Blueberry Wines, Fruit Liquors a nd Grape Wines Similar to the ORAC comparison, the total phenolic val ue of the Southern highbush blueberry wine tested in this study, i.e., 929 mg GAE/L, was used as a benchmark for comparison. For the 26 red wines listed in Table 5 3, the total phenolic values ranged from 700 to 4059 mg GAE/L. A majority of red wines (24/2 6) had total phenols higher than that of the Southern highbush blueberry wine, with only 2 of 26 falling below. The total phenolic contents of the Rose and white wines were all lower than that of the Southern highbush blueberry wine. Of the 8 fruit liquors two had a comparable total phenolic value to that the Southern highbush blueberry wine, while the literature. Listed in Table 5 3 are only 10 red wine types for which th e total anthocyanin contents were reported. Except for the highbush blueberry cultivar Elliot that had a
93 much lower anthocyanin value (i.e., 14.7 mg C3GE/L), most anthocyanin contents of the blueberry wines and grape wines listed in Table 5 3 were comparab le.
94 Table 5 1. The ORAC values, total phenols (TP), anthocyanins and flavonoids of Southern highbush blueberry wine with and without sulfite addition or filtration Values are mean of triplicate measurements. Values are mean of six measurements. Values with the same superscripted letters a re not significantly different ( 0.05). Southern Highbush Blueberry Wine ORAC (mmol TE/L) TP (mg GAE/L) Anthocyanin s ( mg C3GE/L ) Flavonoids (mg CE/L) No sulfite, filtered 23.498.71 a 92952 60.623.51 1233166 Low sulfite, unfiltered 18.543.67 b Average ORAC value = 22.57 0.46 mmol TE/L Sulfite, unfiltered 22.770.46 a Sulfite, filtered 25.485.35 a
95 Table 5 2. Comparison of the ORAC values among the red, Rose, white and blueberry wines Wine ORAC (mmol TE/L) Source Red Wine Graciano 39.9 Davalos and others (2004) Cabernet Sauvignon 34.7 Daval os and others (2004) Tempranillo 30.8 Davalos and others (2004) Aglianico 12.14 Pellegrini (2003) Chianti 11.43 Pellegrini (2003) Sauvignon 8.95 Pellegrini (2003) Aglianico Guardiolo 10.4 12.8 8.4 Fogliano (1999) Fogliano (1999) Solopaca 7.6 Fogliano (1999) Gragnano 7.0 Fogliano (1999) Lacrima Christi Villard Noir Cabernet Sauvignon Tempranillo Montepulciano Sangiovese Merlot Chambourcin Mendocino Pinot Noir Re d Table 6.4 27.20 10.06 21.22 18.94 18.66 17.08 14.67 16.00 9.74 9.52 5.25 Fogliano (1999) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Rose Wine Garnacha 11.2 Davalos and others (2004) Tempranillo 10.0 Davalos and others (2004) Cabernet 8 .95 Davalos and others (2004) Villa Torre 2.42 Pellegrini (2003) Tamerici 2.18 Pellegrini (2003) Bardolino 1.52 Pellegrini (2003) White Wine Albarino 4.84 Davalos and others (2004) Verdejo 3.18 Davalos and others (2004) Vernaccia 1.94 Pellegrini (2003) Pinot 1.68 Pellegrini (2003)
96 Table 5 2. Continued Greco di Tufo 1.61 Pellegrini (2003) Coda di volpe 0.9 Fogliano (1999) Solpaca 0.8 Fogliano (1999) Falanghina 0.8 Fogliano (1999) Lacrima Christi Chardonnay Vidal Blanc Sauvignon Blanc Pinot Grigio 0.6 3.38 5.35 3.33 2.59 2.68 2.28 Fogliano (1999) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Blueberry Wine Southern Highbush, Mixed Highbush, Mixed Highbush, Mixed 22.57 4.5 25.1 16.67 24.39 This study Johnson and others (2011) Sanchez Moreno and others (2003) Highbush, Elliot 18.80 Sanchez Moreno and ot hers (2003) Highbush, Weymouth 9.18 Sanchez Moreno and others (2003)
97 Table 5 3. Total phenol and total anthocyanin comparison among the red, Rose, white and blueberry wines and fruit liquors Wine or Liquor Total Phenols (mg GAE/L) Anthocyanins ( mg C3GE/L ) Source Red Wine Graciano 1468 Davalos and others (2004) Cabernet 1428 Davalos and others (2004) Tempranillo 1302 Davalos and others (2004) Villard Noir 1850 59.02 Sanchez Moreno and others (2003) Cabernet Sauvigno n 1593 1804 59.06 60.23 Sanchez Moreno and others (2003) Tempranillo Montepulciano Sangiovese Merlot Chambourcin 1932 1817 1637 1256 1267 72.33 94.81 52.61 111.70 170.10 Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Sanchez Moreno and others (2003) Aglianico Guardiolo 1300 2300 1400 Fogliano (1999) Fogliano (1999) Solopaca 1200 Fogliano (1999) Gragnano 900 Fogliano (1999) Lacrima Christi Cabernet Sauvignon Me rlot Zinfandel Petite Sirah Pinot Noir Tempranillo Garnacha Cabernet Sauvignon Pinot Noir Enanito Teroldego Cabernet Sauvignon Reference red wine 700 2164 3340 1800 2133 2000 2020 4059 2816 145 5 2446 1277 1530 2358 1037 2529 1963 2063 1663 2352 1390 1600 57 117 255 294 246 443 261 Fogliano (1999) Frankel and others (1995) Frankel and others (1995) Frankel and others (1995) Frankel and others (1995) Frankel and others (1995) Sanchez Moreno and others (2000) Sanchez Moreno and others (2000) Sanchez Moreno and others (2000)
98 Table 5 3. Continued Rigo and others (2000) Rigo and others (2000) Rigo and others (2000) Nyman & Kumpulainen (2001) Heinonen and others (1998) Rose Wine Garnacha 43 2 Davalos and others (2004) Tempranillo 439 Davalos and others (2004) Cabernet Garnacha Tempranillo 389 419 486 330 373 Davalos and others (2004) Sanchez Moreno and others (2000) Sanchez Moreno and others (2000) White Wine A lbarino 214 Davalos and others (2004) Verdejo 186 Davalos and others (2004) Chardonnay 280 306 Sanchez Moreno and others (2003) Vidal Blanc 220 Sanchez Moreno and others (2003) Sauvignon Blanc Pinot Grigio 191 270 191 Sanchez M oreno and others (2003) Sanchez Moreno and others (2003) Coda di volpe 120 Fogliano (1999) Solpaca 150 Fogliano (1999) Falanghina 140 Fogliano (1999) Lacrima Christi Sauvignon Blanc Chardonnay White Zinfandel Malvar Ver dejo Albillo Chardonnay 110 165 193 240 259 243 331 139 265 178 293 201 Fogliano (1999) Frankel and others (1995) Frankel and others (1995) Frankel and others (1995) Sanchez Moreno and
99 Table 5 3. Continued Reference white wine 265 others (2000) Sanchez Moreno and others (2000) Sanchez Moreno and others (2000) Sanchez Moreno and others (2000) Heinonen and others (1998) Blueberry Wine Southern Highbush, Mixed Highbush, Mixed Highbush, Mixed 929 375.4 657. 1 1514 1860 60.62 80.56 162.20 This study Johnson and others (2011) Sanchez Moreno and others (2003) Highbush, Elliot 1470 14.70 Sanchez Moreno and others (2003) Highbush, Weymouth 600 74.69 Sanchez Moreno and others (2003) Rabbiteye (V. ashe i) Fruit Liquor Cranberry Cherry Arctic Bramble Strawberry Rowanberry Cloudberry Cloudberry+Red Raspberry Red Raspberry+Black Current 1150 500 1080 555 610 410 525 545 450 500 415 1050 99.6 Su and Chien (2007) He inonen and others (1998) Heinonen and others (1998) Heinonen and others (1998) Heinonen and others (1998) Heinonen and others (1998) Heinonen and others (1998) Heinonen and others (1998) Heinonen and others (1998)
100 CHAPTER 6 EFFECT OF PULSED UV LIGHT ON SAC C HAROMYCES CEREVI SIAE IN SOUTHERN HIGH BUSH B LUEBERRY WINE AND WH ITE GRAPE WINE Background The growth and metabolism of yeasts ( Saccharomyces cerevisiae ) and bacteria ( Oenococcus oeni ) results in the production of wine, where yeasts ensure alcoholic f ermentation and bacteria ensures malolactic ferment ation (Millet and Lonvaud Funel, 2000). Along with desired microorganisms for fermentation, w ine may also contain acetic acid bacteria, other species of lactic acid bacteria and yeast that produces volatil e acidity, off flavors, and polysaccharides and then affect s wine quality. The undesirable microorganisms in wine include Brettanomyces sp p for yeasts, Acetobacter aceti Pediococcus damnosus and several Lactobacillus sp p for bacteria (Millet and Lonvaud Funel 2000). Beverage industry has been using thermal pasteurization as a mean to produce safe beverages by inactivating pathogens and maintain quality by inactivating spoilage microorganisms. Ten to 20 pasteurization units (PU) are applied to beer for d econtamination of spoilage microorganisms (King and others 1978). However, thermal treat ment has been proven in reducing heat sensitive m icro and macro nutrients and wines which are rich source of polyphenols and antioxidants are believed to have similar de trimental effect on these micronutrients with thermal treatment. It has been the tradition of adding sulfur dioxide (SO 2 ) to prevent microbial spoilage and oxidative browning reactions in wines (Amerine and others 1980). In case of residual sugars in wine, sterile filtration and sorbic acid are used to prevent refermentation of wine. With increase in consumer demand for natural and organic food products, sensitivity for asthmatic consumers, compromising flavor in presence of excess SO 2 and religious
101 restric tions (kosher wine) have always demanded an alternative for wine preservation without addition of sulfur. Heat treatment is applied as an alternative in countries such as Germany, Italy, South Africa and Australia to prevent malolactic fermentation, elimin ate microbial spoilage and browning activity (Somers 1978). During initial stages of wine fermentation, yeasts such as Kloeckera Metschnikowia Candida Hanseniaspora grow in product for a week before Saccharomyces cerevisiae strains dom inate the ferment ation (Cocolin and others 2000 ). The increase in ethanol concentration with the progression of fermentation inhibits the growth of non Saccharomyces yeasts as they are more susceptible to alcohol compared to Saccharomyces spp PUV had shown to be effectiv e in inactivating several microorganisms to ensure food product safety. Kosher wines have thermally pasteurized to ensure safety, but thermal pasteurization has detrimental effects on phytochemicals present in wine. Gram negative bacteria (S. enteritidis ) are less resistant to PUV than Gram positive bacteria ( C cereus ) and fungal spores ( A niger ) are more resistant than E coli and S enteritid i s (Oms Oliu and others 2010). Hence Saccharomyces cerevisiae was selected as a target microorganism for PUV trea tment and was subjected to PUV treatment to observe decontamination effect of PUV. Materials and Methods Materials Southern highbush blueberry wine was obtained from a local blueberry winery Island Grove Wine Company, Hawthorne, FL. The blueberries used to produce the wine were a mixture of several varieties including Star, Windsor, Emerald, Primadonna and
102 Live a Little (South Africa) gra pe wine was used for white wine analysis. Xenon PUV Sterilizer Model: RC 847 (Xenon Corporation, Wilmington, MA) was used for all the experiments. Red Star yeast was used. Experimental P rocedure YPD broth was used as a nutritious liquid growth medium for the propagation of Saccharomyces cerevisiae. It was prepared by dissolving 20 g of bacterio log ical peptone, 10 g of yeast and 20 g of glucose in one liter distilled water followed by autoclaving for 15 min at 121 o C. Five grams of Red Star Montrachet cultu re was grown in 50 ml of YPD broth for 9 h at 30 o C and added to wine. Five m l (2.5 mm thickness) of inoculated blueberry wine was placed in an aluminum dish and subjected to PUV illumination for 12, 14, 16, 18, 20 and 22 s at a distance of 6 cm from the q uartz window of a Xenon PUV sterilizer. Additionally, 10 ml (5 mm thickness) of the sample was treated for 25, 28, 31, 34, 37 and 40 s and 15 ml (7.6 mm thickness) of the sample was treated for 40, 44, 48, 52, 56 and 60 s at the same distance. Ten m l (5 mm thickness) inoculated white wine was treated for 25, 28, 31, 34, 37 and 40 s from the same distance. 3M TM Petrifilm TM yeast and mold count plates (#6407) were used to determine Saccharomyces cerevisiae population before and after PUV treatment. The wine sample was serially diluted in 0.1% peptone water and 1 ml of t he dilution was then plated on P etrifilm TM The plated P etrifilms TM were incubated at 25 o C for 3 to 5 days before counting number of colonies. Ultra violet/visible (UV/VIS) spectrophotometer ( Beckman Coulter, Du 730, Lawrence, KS, USA) was used to read the absorbance of 190 to 1100 nm of light by inoculated wine to understand the possible absorbance of sample during the PUV treatment.
103 Results and Discussion The effects of sample thickness and t reatment time on inactivation of Saccharomyces cerevisiae was investigated in blueberry wine. Temperature When 5 ml of sample was treated with PUV, a log arithmic increase in temperature was observed during initial 10 s of treatment. The temperature was risen by 13.76 o C with 10 s treatment from an initial temperature of 30.67 o C. Further treatment caused a linear increase in temperature. The linear equation s for increase in temperatures of 5 ml 10 ml and 15 ml samples were reported in F igures 6 1, 6 2 and 6 3. Inactivation of Saccharomyces c erevisiae in Blueberry W ine Inoculated blueberry wine was treated with PUV in an aluminum dish at constant distance of 6 cm from quartz window. The same size aluminum dish was used for 5 ml 10 ml and 15 ml samples, which provided a thickness of 2.5 mm 5 mm and 7.6 mm respectively. There was no reduction in Saccharomyces cerevisiae when treated for 10 s for a sample of thickness 2.5 mm (Table 6 1) but a reduction of 1.3 log cfu/ml of Saccharomyces cerevisiae was obs erved with 12 s treatment. However, 22 s treatment resulted i n complete inactivation of 10.7 log cfu/ml Saccharomyces cerevisiae The initial reduction of 0.83 log cfu/ml Saccharomyces cerevisiae was observed with 25 s of PUV treatment for 5.0 mm thick sam ple, where 40 s resu lted in complete inactivation. A 40 s treatment inactivated 0.6 log cfu/ml Saccharomyces cerevisiae and 60 s treatment reduced 9.6 log cfu/ml Saccharomyces cerevisiae As treatment time increases, sample has more exposure to pulsed lig ht and accumulates more energy. But, prolonged treatment times can lead to elevated temperatures that might adversely affect the quality of food product. When blueberries
104 were treated at 3 cm from quartz window for 5 s and 60 s, 1.3 0.6 and 4.9 2.4 log cfu /ml reductions of E. coli O157:H7 respectively were observed, however no significant difference between 5 and 10 s treatments was observed ( Bilakia and Demirci 2007) The mechanism for inactivating microorganisms with PUV treatment is attributed mainly to photochemical, photothermal and photophysical effects. However, photodynamic effects play a vital role in inactivating microorganisms in the sample with photosensitizers. The difference in absorption of UV light by bacteria and surrounded media results in overheating within bacteria and causes bacterial disruption by photothermal mechanism with the exposure of >0.5 J/cm 2 en ergy (Elmnasser and others 2007; 2008). Formation of pyrimidine dimmers including thymine dimmers during UV light proces sing causes ge rmicidal effect of PUV treatment. Dimers cause clonogenic death (inability to replicate) by inhibiting the formation of new DNA chains during cell replication and thus inactivate them. Spore photoproduct 5 thyminyl 5,6 dihydrothymine is formed during UV C processing of bacterial spores. DNA absorbs UV during PUV treatment and results in photochemical effect that plays a major role in inactivation of microorganisms. Single strand breaks and pyrimidine dimmers were formed in yeast cells and damaged DNA during PUV treatment. It was observed that UV induced more DNA damage in yeast cells compared to PUV, however inactivation of yeast cells remained same, suggesting the contribution of other mechanisms than DNA damage (photochemical mechanism) during PUV treatmen t (Gomez Lopez and others 2007). Takeshita and others (2003) observed the effect of continuous wave UV light (CW UV) and PUV on S cerevisiae and concluded the similar DNA damage and formation of
105 single strand breaks and pyrimidine dimmers in yeast cells a fter both CW UV and PUV treatments. Cell membrane damage was evident with increased concentration of eluted protein and structural changes in PUV treated cells. PUV expanded vacuoles and distorted/damaged cell membrane and changed their shape to circular, while CW UV did not alter the yeast cell structure. Photothermal effect can be observed in samples in which temperature of the sample is increased with the availability of higher amount of energy. This heat energy has a synergistic effect in inactivating microorganisms. Saccharomyces cerevisiae can have a D value of 16 min at 51 o C with a Z value of 3.2 o C in grape juice. During the PUV treatment, sample attained a maximum temperature of 42.59 o C, 39.54 o C and 40.77 o C for 5 ml 10 ml and 15 ml to inactiv ate 10.7 10. 7 and 9.6 log cfu/ml Saccharomyces cerevisiae Inactivation of Saccharomyces cerevisiae in White G rape W ine When 10 ml of white wine was treated for decontamination of Saccharomyces cerevisiae at a distance of 6 cm from quartz window, 3. 9 log cfu/ml reduction was observed with a 28 s treatment time (Table 6 2) However, 40 s treatment co uldnot inactivate more than 5.9 log cfu/ml of Saccharomyces cerevisiae in white wine. At the same treatment conditions, PUV was able to inactivate 10.7 lo g cfu/ml of Saccharomyces cerevisiae in blueberry wine. Then samples were measured for pH and the presence of soluble solids. It was observed that blueberry wine had low pH of 3.3 compared to 3.69 of white wine and less soluble solids with 5.8 o Brix compar ed to 7.8 o Brix of white wine. According to Wang and others (2005), Xenon flash lamp treatment at 254 nm for inactivation of E. coli is no different from continuous UV low pressure mercury lamps at same wavelength. PUV treatment at 270nm showed maximum
106 red uction while treatments above 300nm did not inactivate E.coli UV C being the predominant sterilizer in PUV treatment, the UV absorbance of both samples was measured to understand its absorption effect. However, both the samples showed similar UV C absorpt ion at 254 nm as 3.695.
107 Table 6 1. Reducti on of Saccharomyces cerevisiae in blueberry wine when treated with PUV at a distance of 6 cm from quartz window C omplete inactivation of Saccharomyces cerevisiae present in sample. Treatments not 5 ml Blueberry wine 10 ml Blueberry wine 15 ml Blueberry wine Treatment (sec) Log cfu/ml reduction Treatment (sec) Log cfu/ml reduction Treatment (sec) Log cfu/ml reduction 12 1.3 0.8 b 25 0.8 0.5 b 40 0.6 0.2 b 14 2.1 0.3 b 28 2 .0 1.0 b 44 1.3 0. 4 b 16 4.9 0.9 b 31 3.4 0. 5 b 48 2.8 0.7 b 18 6.0 0.4 b 34 5. 4 0. 2 b 52 4.5 1. 2 b 20 9.3 1.7 a 37 6.8 0. 1 b 56 5. 8 1. 2 b 22 10.7 0.2 a 40 10. 7 0.2 a 60 9.6 0. 9 a
108 Tabl e 6 2. Reduction of Saccharomyces cerevisiae in white grape wine when treated with PUV at a distance of 6 cm from quartz window 10 ml Grape White Wine Treatment (sec) Log cfu/ml reduction 28 3.9 0. 2 c 31 4. 3 0.2 bc 34 4.2 0.3 bc 37 5. 1 0. 6 ab 40 5.9 0. 5 a Treatments not connected by same letter are significantly different ( =0.05) from
109 Figure 6 1. Change in surface temperature of 5 ml blueberry wine when treated with PUV for 60 s at a distance of 6 cm from quartz window y = 0.6073x + 7.1702 R = 0.9795 0 5 10 15 20 25 30 35 40 45 50 0 10 20 30 40 50 60 70 Change in Temperature (C) Time (min) 5 ml Wine
110 Figure 6 2. Change in temperature of 10 ml blueberry wine at 2.5 mm distance from surface when treated with PUV for 60 s at a distance of 6 cm from quartz window
11 1 Figure 6 3. Change in temperature of 15 ml bluebe rry wine at 5 mm distance when treated with PUV for 60 s at a distance of 6 cm from quartz window
112 CHAPTER 7 EFFECT OF PULSED UV LIGHT ON P HENOLIC, ANTIOXIDANT ACTIVITY COLOR AND AROMA OF SOUTHERN HIGH BUSH B LUEBERRY WINE Background Secondary metabolit e compounds such as anthocyanins and polyphenols have a major role in the quality of food as they contribute to appearance, taste and healt h benefits (Strack and Wray 1994 ). With higher antioxidant capacity, blueberries and blueberry products have gained m ore interest for the possible health benefits (Lee and others 2002). Several researchers investigated the antioxidant capacity of different Vaccinium sp p but processing effe cts on compositional changes were less investigated. Anthocyanins are one of the main classes of water soluble flavonoids and blueberries are among various fruits and vegetables which are comprised of these anthocyanins. These free radical scavengers interact with bio log ical systems and involve in enzyme inhibiting, antibacterial, card iovascular protection and antioxidant effects. Toxic compounds produced during lipid oxidation trigger harmful reactions and polyphenols can inhibit this lipid oxidation by free radical scavenging, oxygen radical absorbance and chelation of metal ions mech anism and thus prevents harmful reactions initiation. Several factors such as type of product, maturity, variety of product, processing and storage affect the antioxidant capacity of products. The presence of polyphenols including anthocyanins is correlate d with its antioxidant activity in addition to in vitro and in vivo anticancer activity, neuroprotective properties and antiadhesion activity (Schmidt and others 2005). Processing of a blueberry results in the loss of important micro nutrients such as fla vonoids, anthocyanins and polyphenols, and also significantly affects the color. Quinones which were produced during the oxidation of polyphenols in the presence of
113 Polyphenol oxidase (PPO) enzyme can react with anthocyanins to produce brown pigment. Heati ng and addition of SO 2 were proved to be effective in inhibiting PPO enzyme activity. Phenolic compounds were reduced when frozen high bush blueberries were processed and concentrated (Skrede and others 2000 ). Thawing, crushing, depectinization, and pressi ng steps of juice and concentrate processing resulted in significant deteriorati on of anthocyanins, polyphenol s and antioxidant activity (Lee 2002). According to Schmidt (2005), although heat processing of blueberry products may not show significant differ ence in antioxidant activity and total phenolic content in comparison with fresh and frozen fruit, it can result in lower bioactivities. The study indicated lower antiproliferative activity in products that retained total phenols and antioxidant activity. Processing effects on antioxidant capacity of blueberry food products were evaluated at different temperatures and pH. Extraction at 60 o C retained higher anthocyanin and antioxidant capacity in comparison with extraction at 25 o C, but significantly reduced durin g storage at room temperature. Puree extracted in pH 4 and 7 had lower antioxidant capacity compared to the puree extracted in pH 1 ( Kalt and Dufour 1997 ). PUV as a nonthermal pasteurizer gained importance in food industry which can inactivate spoil age microorganisms and pathogens at very short processing times. Human health enhancing chemical substances such as scoparone in grapes, 6 methoxymellein in carrots, resveratrol in grapes and anthocyanins in strawberries and apples can be induced using UV treatment. PUV treatment at 30 J/cm 2 energy had not affect ed protein, nitrosamin e, benzopyrene and vitamin C in frankfurters compared to untreated (Oms Oliu and others 2010). PUV illumination on southern high bush
114 blueberries enhanced their antioxidant cap acity, phytochemicals and antioxidant enzyme activity while maintaining their other quality characteristics such as color, firmness and soluble solids (Guner 2012). The objective of this study is to analyze the effect of PUV illumination on phenolics, anti oxidants, color and aroma of southern high bush blueberry wine. Materials and Methods Southern Highb ush Blueberry Wine Samples Southern highbush blueberry wine was obtained from a local blueberry winery Island Grove Wine Company, Hawthorne, FL. The bluebe rries used to produce the wine were a mixture of several varieties incl uding Star, Windsor, Emerald, Primadonna and Determination o f Antioxidant Capacity Antioxidant capacity of the Southern highbush blueb erry wine was measured by the Oxygen Radical Absorbance Capacity (ORAC) method. The ORAC values were expressed as mmol Trolox equivalent (TE) per liter Modified method per Huang and others (2002), 50 L ORAC Phosphate Buffer (75 mM, ORAC PB) and appropria tely diluted samples wer e added to a 96 well black plate (Fisher Scientific). This was followed by addition of 100 L, 20 nM fluorescein working solution to all filled wells. The mixture was incubated at 37 o C for 10 min before the addition of peroxyl radi cal amidinopropane) dihydrochloride (140 mM, AAPH). The rate of fluorescence decay was monitored over time by calculating the area under the fluorescent decay curve and quantified using a standard curve of TROLOX, using a Spectra Ma x Gemini XPS microplate reader (Molecular Devices, Sunnyvale, CA). The
115 fluorescence was monitored at 485 nm excitation and 530 nm emissions for 40 min at 1 min intervals. The antioxidant capacities of the extracts were expressed as mmol TE/L of blueberry wine. For the other wines, the ORAC procedure was basically the same as above, except for different dilutions as: 1:1000, 1:1500, 1:2000, and 1:3000. Determination o f Total Phenols Total phenolics were determined using a modification of Fo lin ethod. To a 96 well clear plate (Fisher Scientific, Pittsburg, PA), 12.5 l of 2 N Folin phenol reagent was added to 50 l of deionized distilled water (ddH 2 O) and 12.5 l of wine sample. After 5 min, 7% sodium carbonate (Na 2 CO 3 ) solution (125 l) was added to the mixture and incubated (90 min, 25 o C). The absorbance of the sample was measured at 750 nm versus a reagent blank using a microplate reader. A standard curve for total phenolics was developed using gallic acid. The concentration was exp ressed as mg of gallic acid equivalents (GAE)/L of wine. Determination o f Total Flavonoids A standard colorimetric assay (Kim and others 2003) with slight modifications was used to qua ntify total flavonoid content. To a 96 well clear plate, 25 l of the w ine sample was added to 125 l of ddH 2 O. Subsequently, 7.5 l of 5% sodium nitrate (NaNO 2) was added to the mixture and allowed to stand for 5 min. Fifteen microliters of 10% aluminum chloride (AlCl 3 ) was added to the mixture and incubated at ambient tempe rature for an additional 5 min. Following that, 50 l of sodium hydroxide (1 M, NaOH) were added to the mixture and immediately diluted by the addition of 27.5l of ddH 2 O. The absorbance of the mixture was measured at 510 nm against a reagent blank and com pared to a catechin standard using a micro plate reader. The total flavonoids was expressed as mg of catechin equivalents (CE)/L of wine.
116 Determination o f Total Anthocyanins Total anthocyanins were determined by the pH differential method (Benvenuti and ot hers 2004). Two buffer systems, potassium chloride (KCl) (pH 1.0, 0.025 M) and sodium acetate (NaC 2 H 3 O 2 ) (pH 4.5, 0.4 M) were utilized. An aliquot of the blueberry wine was diluted (1:10) and adjusted to pH 1.0 and pH 4.5 using the prepared buffers. Subseq uently, the solutions were incubated at ambient temperature for 20 min. Absorbance was measured using a UV/VIS spectrophotometer (Beckman Coulter, Du 730, Life Sciences UV/VIS, Lawrence, KS) at 510 nm and 700 nm at each pH, respectively. Results were calcu lated using E quation 7 1, 7 2 and expressed as mg of cyanidin 3 glucoside/L of wine. A = (A 510nm A 700nm ) pH1.0 (A 510nm A 700nm ) pH4.5 ( 7 1 ) Anthocyanins = A ( 7 2 ) absorptivity [26, 900] (Sellappan and others 2002). Color Color machine vision system (CMVS) (University of Florida, Gainesvil le, FL, USA) was used to measure the color of treated and untreated blueberry wine. It consists of a Nikon D200 digital color camera (Nikon Corp, Japan), housed in a light box [42.5 cm (Width) x 61.0 cm (Length) x 78.1 cm (Height)] (Wallat and others 2002) The image was captured using the camera (focal light, 35 mm; polarization, 18.44 mm) that was connected to a computer prior to color analysis. Values of L (lightness), a* (redness) and b* (yellowness) were analyzed using LensEye software (Engineering and Cybersolutions Inc. Gainesville, FL, USA). The camera was calibrated with a standard
117 red tile (L: 48.62; a*: 49.04, b*: 25.72) (Labsphere, North Sutton, NH, USA). The results were reported as the hue using the Equation 7 3 Hue=tan 1 [b*/a*] ( 7 3 ) Gas Chromatography Mass Spect rometry Volatile Aroma Analysis Hewlett Packard 5973 mass selective detector, HP 7683 series injector and HP 6890 series GC system was used for analysis. PUV treated and untreated wine samples were analyzed f or aroma volatiles using GC MS. Briefly, 10 ml of wine in a 40 ml glass vial with a silicone/PTFE septa screw cap was equilibrated at room temperature for 20 min, followed by a 30 min extraction using Static Head Space Solid Phase Micro Extraction (HS SPME ) at room temperature with gentle magnetic stir bar stirring. The column used for analysis was DB WAX 30 m length, 0.32 mm internal diameter and 0.25 m film thickness. It had a delay time of 40 min and ended at 25 min. The initial GC oven temperature was 40 o C with a 2 min hold and the injector port was held at 220 o C. The temperature was increased at 7.0 o C per min to 240 o C, where it was held for 9.5 min. GC MS identifications were made by analyzing mass spectra data using two libraries NIST 98 and W8 Results and Discussion T otal Pheno lics, Anthocyanins, Flavonoids a nd Antioxidant Capacity Table 7 1 shows the total phenolics, anthocyanins, flavonoids and antioxidant capacity (ORAC) in control and PUV treated blueberry wine. There was no significant difference ( ) in control and PUV treated samples till the tested 30 s treatment. However, Table 7 2 shows the significant decrease in total phenolics, anthocyanins, flavonoids and antioxidant capacity (ORAC) of blu eberry wine with heat treatment. An tioxidant capacity of plant tissues depends on anthocyanins and phenolic compounds
118 present in it (Wang 2009). The results from this study indicated no significant change in total phenolics and anthocyanins of PUV treated blueberry wine and these results we re substantiated with antioxidant capacity measured through ORAC with no significant change after treatment. The nonthermal range of PUV with 10.9 o C rise from initial temperature for the 30 s treatment proved the minimal or negligible e ffect of PUV treatm ent on micro nutrients such as anthocyanins and polyphenols. UV light can produce oxygen radicals at 185 to 195 nm during UV light processing which can be formed into ozone and cause off flavors in food (Krishnamurthy 2006). A strong photo sensitizer can ab sorb visible and UV light and transfers this energy into highly reactive singlet oxygen. The counter mechanisms for the function of singlet oxygen can lead to the consumption of some antioxidants in sample and there by reduces the antioxidant capacity of t he sample. However, no effect on antioxidant capacity and absence of a strong photo sensitizer in blueberry wine suggests any oxygen radical production at low treatment times in blueberry wine. Yeom and others (2000) reported that orange juice had lost fla vor compounds and vitamin C during the thermal processing at 94 o C for 30 s and at the end of 15 d at 4 o C Degenerative disease can be prevented with the consumption of citrus juices especially orange juice which has higher antioxidant, polyphenol, carote noid and vitamin C content. Undesirable reactions such as nutrient loss and nonenzymatic browning can occur during thermal processing and trigger degradation of vitamin C during nonenzymatic browning (Roig and others 1999). Heat treatment affected total ph enolic content and color of pomegranate juice compared to unpasteurized juice (Alper and others 2005). Pozo Insfran and others (2006) observed a reduction of 16% anthocyanin,
119 26% soluble phenolics, 10% antioxidant capacity with a significant difference in color, flavor, aroma and overall likability after thermal pasteurization of muscadine grape juice. Our study confirmed the similar trend in reduction of these micronutrients in blueberry wine when treated at 65, 70 and 75 o C. Although, PUV treatment contri buted to a rise in temperature (to 42 o C) it was much lower than pasteurization temperatures and did not adversely affect these micronutrients in PUV processed blueberry wine. Color Color was measured interms of L*a*b values, where L* = 100 is t he co ordinate of the color white, L* = 0 for black according to the Commission Internationale de quality factor (Batu 2004) with a* value as an indicator of red color and b* value for yellow. Hue is normally expressed in degrees () and in general 0 (Red), 90 (Yellow), 180 (Green) and 270 (blue). Table 7 2 shows the color (hue) and lightness values of the CIELAB system after PUV treatments. There was no significant ( =0.05 ) difference observed in the treated samples and control for hue and lightness. Anthocyanins are largely responsible for the color of blueberry wine. The rapid PUV treatment at lower temperature did not show any significant difference in anthocyanins Hence, the color of the sample was not affected and is confirmed with no significant difference in the hue and lightness values of PUV treated and untreated blueberry wine. Volatile Aroma The major compounds identified using libraries are acetic acid ethyl ester, isoamyl alcohol, hexanoic acid ethyl ester, octanoic acid ethyl ester, 2,4 hexadienoic acid ethyl ester, decanoid acid ethyl ester, phenethyl alcohol and butanedioic acid ethyl ester
120 (Figure 7 1, 7 2) No new compound was identified in PUV tre ated samples. Oxidation of methionine produces dimethyl disulfide and secondary oxidation products of lipid oxidation such as hexanal and pentanal are major light induced off flavors in milk (Mestdagh and others 2005). Still and sparkling wines were expose d to fluorescent light to observe the effect of light on aroma of wine p acked in green and flint (c;ear) bottles. A significant decrease in aroma of still and sparkling wines was produced after an exposure for 3.4 h and 3.3 h in flint glass, 18 h and 31.1 h in green bottles. A decrease in citrus aroma intensity and increase in cooke d cabbage, corn nuts, wet dog/ wet wool and soy or marmite intensity was observ ed in descriptive analysis (Doze n and Noble 1989). Lipid peroxidation produces several potent aldehy des in presence of oxygen and light and thus develops off flavors. A short treatment time in PUV processing avoids these reactions. Oxidized or cooked off flavors were observed in orange juice when exposed to light in presence of oxygen; however no off fla vor was observed in absence of oxygen. Esters provide a fruity top note in fresh juices and are often observed to be degraded during thermal processing (P e rez Cacho and Rousef 2008). The identified esters in sample may provide the fruity top note to blueb erry wine. An increase and decrease of carotenoids in grapes before verasion and during maturity in comparison to shaded grapes suggests the significant effect of light in diminishing carotenoids after ripening. Photo carotene was proposed to be one possible pathway for ionone in grapes (Mendes Pinto 2009). In the current study, there was no significant effect of PUV processing on polyphenols, anthocyanins, color and aroma. This suggests the possible absence of photo oxid ation at the tested conditions.
121 Figure 7 1. GC MS chromatogram for volatile aroma of 5 s PUV treated blueberry wine
122 Figure 7 2. GC MS chromatogram for volatile aroma of 25 s PUV treated blueberry wine
123 Table 7 1. Effect of PUV on ORA C, total phenolics, anthocyanins and flavonoids in blueberry wine when treated at a distance of 6 cm from quartz window Treatments not connected by same letter are significantly different ( =0.05) from
124 Table 7 1 Effect of PUV treatment on lightness and hue of blueberry wine when treated at a distance of 6 cm f rom quartz window Treatments not connected by same letter are sign ificantly different ( =0.05) from
125 Table 7 2. Identified major volatile aroma compounds of blueberry wine when treated at a distance of 6 cm from quartz window Retention Time (min) Compound 1.9 Ethyl acetate 6.7 Isoamyl alcohol 7.09 Ethyl hexanoate 10.9 Ethyl octanoate 11.72 2,4 hexadienoic acid 14.42 Ethyl decanoate 15.04 Butanedioic acid 18.68 Phenythyl alcohol
126 CHAPTER 8 CONCLUSIONS AND RECO MMENDATIONS Results from this study indicated that PUV treatments were effective in reducing the antigenicity of i solated milk and egg proteins. Degree of reduction was dependent of treatment time, as longer treatment time resulted in higher energy absorption by sample during the PUV treatments. SDS PAGE showed the formation of a new band at 10 kDa for whey protein an d the intensity of band was found to be increased with increase in treatment time suggesting the possible fragmentation of protein. The aggregated protein with PUV treatment did not penetrate through gel and was observed at the entrance. Higher reduction o casein antigenicity was found to be at 180s treatment time at a distance of 10.2 cm from UV source. HHP treatment at 600 MPa for 5, 15, and 30 min at three initial temperatures of 4, 21, and 70 o C increased the antigenicity of isolated eg g proteins, suggesting the avoidance of the techno log y in developing hypo allergic egg products. PUV inactivated 10. 7 10. 7 and 9.6 log cfu/ml of Saccharomyces cerevisiae in inoculated blueberry wine at a distance of 6 cm from quartz window at 22, 40 and 60 s treatments respectively. 1. 3 0.8 and 0.6 log cfu/ml reduction of Saccharomyces cerevisiae was observed in 5, 10 and 15 ml of samples treated for 12, 25 and 40 s respectively. PUV inactivated onl y 5.9 log cfu/ml of Saccharomyces cerevisiae in white w ine, whereas 10. 7 log cfu/ml of Saccharomyces cerevisiae was observed in red wine. Less pH and soluble solids in red wine compared to white wine might have contributed for higher reduction with PUV treatment. Photo thermal, photo physical and photo chemica l mechanisms were suggested by researchers for the inactivation of microorganisms by PUV. N o significant difference was observed in ORAC, total
127 phenolic content, flavonoids, anthocyanins, lightness and hue of PUV treated and untreated blueberry wine, sugge sting no detrimental effect of PUV treatment on these micro nutrients. Ethyl acetate, isoamyl alcohol, ethyl hexanoate, ethyl octanoate, 2,4 hexadienoic acid ethyl ester, ethyl decanoate, phenethyl alcohol and butanedioic acid ethyl ester were major aroma c ompounds identified in treated and untreated blueberry wine using GC MS analysis. No new peaks for volatile aroma in PUV treated blueberry wine were produced, suggesting the possible retain of aroma in blueberry wine with PUV treatment. A significant diffe rence was observed in ORAC, total phenolics, flavonoids and anthocyanins of thermally treated and untreated blueberry wine samples. Based on our results and literature, it can be deduced that PUV may be utilized for microbial and allergen inactivation, wh ile preserving the quality of food matrix. HHP increased antigenicity of isolated egg proteins at the tested conditions. The mechanism involved in the reduction of allergens using PUV need to be investigated for better understanding of the process. The PUV efficacy in reducing the antigenicity of these proteins needs to be confirmed with clinical trials. PUV has a potential to be an alternative food processing techno log y for inactivation of microorganisms without having any detrimental effect on micro nutrie nts such as polyphenols and antioxidants while preserving the volatile aroma.
128 APPENDIX NOMENCLATURE % Percentage C Degree celsius A Absorbance of analyte a* Redness Atm Atmosphere b* Yellowness C Carbon CaOH Calcium hydroxide D Dilution factor ddH 2 O Dionized distilled water g Gram h Hour K Kelvin Kg Kilogram Kg/h Kilogram per hour KHz Kilohertz KJ Kilojoule per hour KJ/Kg Kilojoule per kilogram kPa Kilopascals KWH Kilowatt hour L* Lightness mg Milligram
129 MHz Megahertz mL Millilite r mm Millimeter MPa Megapascals N Normality NaCL Sodium chloride NaOH Sodium hydroxide nm Nanometer ns Nanosecond Tan 1 Inversed tangent V Volume of titrant Micrometer
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143 BIOGRAPHICAL SKETCH Akshay Kumar Anugu is a native of India In 2006, h e graduated with h is b egree in f ood t echn o log y from Osmania Univer sity in India Then, he had enrolled into a m program in f ood s cience at Food and Animal S cience s Department in Alabama A griculture & M echanical University and graduated in 2009. A nugu was offered a Graduate Assistantship in Fall 2009 t o pursue hi s d octoral studies at Un iversity of Florida Gainesville, FL During h is tenure, he served as the Graduate Student Representative of the F ood S cience and Human Nutrition D epartment for Graduate Assistant United He is an active me mber of several nonprofit organizations such as Asha for Ed ucation and NRI Samay and other organizations such as India Against Corruption and Aam Aadmi Party.